Methods and Systems for Processing Polynucleotides

ABSTRACT

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/717,871, filed Sep. 27, 2017, which is a continuation-in-part of U.S.patent application Ser. No. 14/752,641, filed Jun. 26, 2015, whichclaims priority to U.S. Provisional Patent Application No. 62/061,567,filed Oct. 8, 2014, and U.S. Provisional Patent Application No.62/017,558, filed Jun. 26, 2014, each of which application is entirelyincorporated herein by reference.

BACKGROUND

Significant advances in analyzing and characterizing biological andbiochemical materials and systems have led to unprecedented advances inunderstanding the mechanisms of life, health, disease and treatment.Among these advances, technologies that target and characterize thegenomic make up of biological systems have yielded some of the mostgroundbreaking results, including advances in the use and exploitationof genetic amplification technologies, and nucleic acid sequencingtechnologies.

Nucleic acid sequencing can be used to obtain information in a widevariety of biomedical contexts, including diagnostics, prognostics,biotechnology, and forensic biology. Sequencing may involve basicmethods including Maxam-Gilbert sequencing and chain-terminationmethods, or de novo sequencing methods including shotgun sequencing andbridge PCR, or next-generation methods including polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrentsemiconductor sequencing, HeliScope single molecule sequencing, SMRT®sequencing, and others.

Despite these advances in biological characterization, many challengesstill remain unaddressed, or relatively poorly addressed by thesolutions currently being offered. The present disclosure provides novelsolutions and approaches to addressing many of the shortcomings ofexisting technologies.

BRIEF SUMMARY

Provided herein are methods, compositions and systems for analyzingindividual cells or small populations of cells, including the analysisand attribution of nucleic acids from and to these individual cells orcell populations.

An aspect of the disclosure provides a method of analyzing nucleic acidsfrom cells that includes providing nucleic acids derived from anindividual cell into a discrete partition; generating one or more firstnucleic acid sequences derived from the nucleic acids within thediscrete partition, which one or more first nucleic acid sequences haveattached thereto oligonucleotides that comprise a common nucleic acidbarcode sequence; generating a characterization of the one or more firstnucleic acid sequences or one or more second nucleic acid sequencesderived from the one or more first nucleic acid sequences, which one ormore second nucleic acid sequences comprise the common barcode sequence;and identifying the one or more first nucleic acid sequences or one ormore second nucleic acid sequences as being derived from the individualcell based, at least in part, upon a presence of the common nucleic acidbarcode sequence in the generated characterization.

In some embodiments, the discrete partition is a discrete droplet. Insome embodiments, the oligonucleotides are co-partitioned with thenucleic acids derived from the individual cell into the discretepartition. In some embodiments, at least 10,000, at least 100,000 or atleast 500,000 of the oligonucleotides are co-partitioned with thenucleic acids derived from the individual cell into the discretepartition.

In some embodiments, the oligonucleotides are provided attached to abead, where each oligonucleotide on a bead comprises the same barcodesequence, and the bead is co-partitioned with the individual cell intothe discrete partition. In some embodiments, the oligonucleotides arereleasably attached to the bead. In some embodiments, the bead comprisesa degradable bead. In some embodiments, prior to or during generatingthe one or more first nucleic acid sequences the method includesreleasing the oligonucleotides from the bead via degradation of thebead. In some embodiments, prior to generating the characterization, themethod includes releasing the one or more first nucleic acid sequencesfrom the discrete partition.

In some embodiments, generating the characterization comprisessequencing the one or more first nucleic acid sequences or the one ormore second nucleic acid sequences. The method may also includeassembling a contiguous nucleic acid sequence for at least a portion ofa genome of the individual cell from sequences of the one or more firstnucleic acid sequences or the one or more second nucleic acid sequences.Moreover, the method may also include characterizing the individual cellbased upon the nucleic acid sequence for at least a portion of thegenome of the individual cell.

In some embodiments, the nucleic acids are released from the individualcell in the discrete partition. In some embodiments, the nucleic acidscomprise ribonucleic acid (RNA), such as, for example, messenger RNA(mRNA). In some embodiments, generating one or more first nucleic acidsequences includes subjecting the nucleic acids to reverse transcriptionunder conditions that yield the one or more first nucleic acidsequences. In some embodiments, the reverse transcription occurs in thediscrete partition. In some embodiments, the oligonucleotides areprovided in the discrete partition and include a poly-T sequence. Insome embodiments, the reverse transcription comprises hybridizing thepoly-T sequence to at least a portion of each of the nucleic acids andextending the poly-T sequence in template directed fashion. In someembodiments, the oligonucleotides include an anchoring sequence thatfacilitates hybridization of the poly-T sequence. In some embodiments,the oligonucleotides include a random priming sequence that can be, forexample, a random hexamer. In some embodiments, the reversetranscription comprises hybridizing the random priming sequence to atleast a portion of each of the nucleic acids and extending the randompriming sequence in template directed fashion.

In some embodiments, a given one of the one or more first nucleic acidsequences has sequence complementarity to at least a portion of a givenone of the nucleic acids. In some embodiments, the discrete partition atmost includes the individual cell among a plurality of cells. In someembodiments, the oligonucleotides include a unique molecular sequencesegment. In some embodiments, the method can include identifying anindividual nucleic acid sequence of the one or more first nucleic acidsequences or of the one or more second nucleic acid sequences as derivedfrom a given nucleic acid of the nucleic acids based, at least in part,upon a presence of the unique molecular sequence segment. In someembodiments, the method includes determining an amount of the givennucleic acid based upon a presence of the unique molecular sequencesegment.

In some embodiments, the method includes, prior to generating thecharacterization, adding one or more additional sequences to the one ormore first nucleic acid sequences to generate the one or more secondnucleic acid sequences. In some embodiments, the method includes addinga first additional nucleic acid sequence to the one or more firstnucleic acid sequences with the aid of a switch oligonucleotide. In someembodiments, the switch oligonucleotide hybridizes to at least a portionof the one or more first nucleic acid sequences and is extended in atemplate directed fashion to couple the first additional nucleic acidsequence to the one or more first nucleic acid sequences. In someembodiments, the method includes amplifying the one of more firstnucleic acid sequences coupled to the first additional nucleic acidsequence. In some embodiments, the amplifying occurs in the discretepartition. In some embodiments, the amplifying occurs after releasingthe one or more first nucleic acid sequences coupled to the firstadditional nucleic acid sequence from the discrete partition.

In some embodiments, after the amplifying, the method includes addingone or more second additional nucleic acid sequences to the one or morefirst nucleic acid sequences coupled to the first additional sequence togenerate the one or more second nucleic acid sequences. In someembodiments, the adding the one or more second additional sequencesincludes removing a portion of each of the one or more first nucleicacid sequences coupled to the first additional nucleic acid sequence andcoupling thereto the one or more second additional nucleic acidsequences. In some embodiments, the removing is completed via shearingof the one or more first nucleic acid sequences coupled (e.g., ligated)to the first additional nucleic acid sequence.

In some embodiments, prior to generating the characterization, themethod includes subjecting the one or more first nucleic acid sequencesto transcription to generate one or more RNA fragments. In someembodiments, the transcription occurs after releasing the one or morefirst nucleic acid sequences from the discrete partition. In someembodiments, the oligonucleotides include a T7 promoter sequence. Insome embodiments, prior to generating the characterization, the methodincludes removing a portion of each of the one or more RNA sequences andcoupling an additional sequence to the one or more RNA sequences. Insome embodiments, prior to generating the characterization, the methodincludes subjecting the one or more RNA sequences coupled to theadditional sequence to reverse transcription to generate the one or moresecond nucleic acid sequences. In some embodiments, prior to generatingthe characterization, the method includes amplifying the one or moresecond nucleic acid sequences. In some embodiments, prior to generatingthe characterization, the method includes subjecting the one or more RNAsequences to reverse transcription to generate one or more DNAsequences. In some embodiments, prior to generating thecharacterization, the method includes removing a portion of each of theone or more DNA sequences and coupling one or more additional sequencesto the one or more DNA sequences to generate the one or more secondnucleic acid sequences. In some embodiments, prior to generating thecharacterization, the method includes amplifying the one or more secondnucleic acid sequences.

In some embodiments, the nucleic acids include complementary (cDNA)generated from reverse transcription of RNA from the individual cell. Insome embodiments, the oligonucleotides include a priming sequence andare provided in the discrete partition. In some embodiments, the primingsequence includes a random N-mer. In some embodiments, generating theone or more first nucleic acid sequences includes hybridizing thepriming sequence to the cDNA and extending the priming sequence intemplate directed fashion.

In some embodiments, the discrete partition includes switcholigonucleotides comprising a complement sequence of theoligonucleotides. In some embodiments, generating the one or more firstnucleic acid sequences includes hybridizing the switch oligonucleotidesto at least a portion of nucleic acid fragments derived from the nucleicacids and extending the switch oligonucleotides in template directedfashion. In some embodiments, generating the one or more first nucleicacid sequences includes attaching the oligonucleotides to the one ormore first nucleic acid sequences. In some embodiments, the one or morefirst nucleic acid sequences are nucleic acid fragments derived from thenucleic acids. In some embodiments, generating the one or more firstnucleic acid sequences includes coupling (e.g., ligating) theoligonucleotides to the nucleic acids.

In some embodiments, a plurality of partitions comprises the discretepartition. In some embodiments, the plurality of partitions, on average,comprises less than one cell per partition. In some embodiments, lessthan 25% of partitions of the plurality of partitions do not comprise acell. In some embodiments, the plurality of partitions comprisesdiscrete partitions each having at least one partitioned cell. In someembodiments, fewer than 25%, fewer than 20%, fewer than 15%, fewer than10%, fewer than 5% or fewer than 1% of the discrete partitions comprisemore than one cell. In some embodiments, at least a subset of thediscrete partitions comprises a bead. In some embodiments, at least 75%,at least 80%, at least 85%, at least 90%, at least 95% or at least 99%of the discrete partitions comprise at least one cell and at least onebead. In some embodiments, the discrete partitions include partitionednucleic acid barcode sequences. In some embodiments, the discretepartitions include at least 1,000, at least 10,000, or at least 100,000different partitioned nucleic acid barcode sequences. In someembodiments, the plurality of partitions comprises at least 1,000, atleast 10,000 or at least 100,000 partitions.

In another aspect, the disclosure provides a method of characterizingcells in a population of a plurality of different cell types thatincludes providing nucleic acids from individual cells in the populationinto discrete partitions; attaching oligonucleotides that comprise acommon nucleic acid barcode sequence to one or more fragments of thenucleic acids from the individual cells within the discrete partitions,where a plurality of different partitions comprise different commonnucleic acid barcode sequences; and characterizing the one or morefragments of the nucleic acids from the plurality of discretepartitions, and attributing the one or more fragments to individualcells based, at least in part, upon the presence of a common barcodesequence; and characterizing a plurality of individual cells in thepopulation based upon the characterization of the one or more fragmentsin the plurality of discrete partitions.

In some embodiments, the method includes fragmenting the nucleic acids.In some embodiments, the discrete partitions are droplets. In someembodiments, the characterizing the one or more fragments of the nucleicacids includes sequencing ribosomal deoxyribonucleic acid from theindividual cells, and the characterizing the cells comprises identifyinga cell genus, species, strain or variant. In some embodiments, theindividual cells are derived from a microbiome sample. In someembodiments, the individual cells are derived from a human tissuesample. In some embodiments, the individual cells are derived fromcirculating cells in a mammal. In some embodiments, the individual cellsare derived from a forensic sample. In some embodiments, the nucleicacids are released from the individual cells in the discrete partitions.

An additional aspect of the disclosure provides a method ofcharacterizing an individual cell or population of cells that includesincubating a cell with a plurality of different cell surface featurebinding group types, where each different cell surface binding grouptype is capable of binding to a different cell surface feature, andwhere each different cell surface binding group type comprises areporter oligonucleotide associated therewith, under conditions thatallow binding between one or more cell surface feature binding groupsand its respective cell surface feature, if present; partitioning thecell into a partition that comprises a plurality of oligonucleotidescomprising a barcode sequence; attaching the barcode sequence tooligonucleotide reporter groups present in the partition; sequencing theoligonucleotide reporter groups and attached barcodes; andcharacterizing cell surface features present on the cell based uponreporter oligonucleotides that are sequenced.

An additional aspect of the disclosure provides a composition comprisinga plurality of partitions, each of the plurality of partitionscomprising an individual cell and a population of oligonucleotides thatcomprise a common nucleic acid barcode sequence. In some embodiments,the plurality of partitions comprises droplets in an emulsion. In someembodiments, the population of oligonucleotides within each of theplurality of partitions is coupled to a bead disposed within each of theplurality of partitions. In some embodiments, the individual cell hasassociated therewith a plurality of different cell surface featurebinding groups associated with their respective cell surface featuresand each different type of cell surface feature binding group includesan oligonucleotide reporter group comprising a different nucleotidesequence. In some embodiments, the plurality of different cell surfacefeature binding groups includes a plurality of different antibodies orantibody fragments having a binding affinity for a plurality ofdifferent cell surface features.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in the art from the following detaileddescription, wherein only illustrative embodiments of the presentdisclosure are shown and described. As will be realized, the presentdisclosure is capable of other and different embodiments, and itsseveral details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 schematically illustrates a microfluidic channel structure forpartitioning individual or small groups of cells.

FIG. 2 schematically illustrates a microfluidic channel structure forco-partitioning cells and beads or microcapsules comprising additionalreagents.

FIG. 3 schematically illustrates an example process for amplificationand barcoding of cell's nucleic acids.

FIG. 4 provides a schematic illustration of use of barcoding of cell'snucleic acids in attributing sequence data to individual cells or groupsof cells for use in their characterization.

FIG. 5 provides a schematic illustrating cells associated with labeledcell-binding ligands.

FIG. 6 provides a schematic illustration of an example workflow forperforming RNA analysis using the methods described herein.

FIG. 7 provides a schematic illustration of an example barcodedoligonucleotide structure for use in analysis of ribonucleic (RNA) usingthe methods described herein.

FIG. 8 provides an image of individual cells co-partitioned along withindividual barcode bearing beads

FIG. 9A-E provides schematic illustration of example barcodedoligonucleotide structures for use in analysis of RNA and exampleoperations for performing RNA analysis.

FIG. 10 provides schematic illustration of example barcodedoligonucleotide structure for use in example analysis of RNA and use ofa sequence for in vitro transcription.

FIG. 11 provides schematic illustration of an example barcodedoligonucleotide structure for use in analysis of RNA and exampleoperations for performing RNA analysis.

FIG. 12A-B provides schematic illustration of example barcodedoligonucleotide structure for use in analysis of RNA.

FIG. 13A-C provides illustrations of example yields from template switchreverse transcription and PCR in partitions.

FIG. 14A-B provides illustrations of example yields from reversetranscription and cDNA amplification in partitions with various cellnumbers.

FIG. 15 provides an illustration of example yields from cDNA synthesisand real-time quantitative PCR at various input cell concentrations andalso the effect of varying primer concentration on yield at a fixed cellinput concentration.

FIG. 16 provides an illustration of example yields from in vitrotranscription.

FIG. 17 shows an example computer control system that is programmed orotherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

I. Single Cell Analysis

Advanced nucleic acid sequencing technologies have yielded monumentalresults in sequencing biological materials, including providingsubstantial sequence information on individual organisms, and relativelypure biological samples. However, these systems have not proveneffective at being able to identify and characterize sub-populations ofcells in biological samples that may represent a smaller minority of theoverall make up of the sample, but for which individualized sequenceinformation could prove even more valuable.

Most nucleic acid sequencing technologies derive the nucleic acids thatthey sequence from collections of cells derived from tissue or othersamples. The cells can be processed, en masse, to extract the geneticmaterial that represents an average of the population of cells, whichcan then be processed into sequencing ready DNA libraries that areconfigured for a given sequencing technology. As will be appreciated,although often discussed in terms of DNA or nucleic acids, the nucleicacids derived from the cells may include DNA, or RNA, including, e.g.,mRNA, total RNA, or the like, that may be processed to produce cDNA forsequencing, e.g., using any of a variety of RNA-seq methods. Followingfrom this processing, absent a cell specific marker, attribution ofgenetic material as being contributed by a subset of cells or all cellsin a sample is virtually impossible in such an ensemble approach.

In addition to the inability to attribute characteristics to particularsubsets of populations of cells, such ensemble sample preparationmethods also are, from the outset, predisposed to primarily identifyingand characterizing the majority constituents in the sample of cells, andare not designed to be able to pick out the minority constituents, e.g.,genetic material contributed by one cell, a few cells, or a smallpercentage of total cells in the sample. Likewise, where analyzingexpression levels, e.g., of mRNA, an ensemble approach would bepredisposed to presenting potentially grossly inaccurate data from cellpopulations that are non-homogeneous in terms of expression levels. Insome cases, where expression is high in a small minority of the cells inan analyzed population, and absent in the majority of the cells of thepopulation, an ensemble method would indicate low level expression forthe entire population.

This original majority bias is further magnified, and even overwhelming,through processing operations used in building up the sequencinglibraries from these samples. In particular, most next generationsequencing technologies rely upon the geometric amplification of nucleicacid fragments, such as the polymerase chain reaction, in order toproduce sufficient DNA for the sequencing library. However, suchgeometric amplification is biased toward amplification of majorityconstituents in a sample, and may not preserve the starting ratios ofsuch minority and majority components. By way of example, if a sampleincludes 95% DNA from a particular cell type in a sample, e.g., hosttissue cells, and 5% DNA from another cell type, e.g., cancer cells, PCRbased amplification can preferentially amplify the majority DNA in placeof the minority DNA, both as a function of comparative exponentialamplification (the repeated doubling of the higher concentration quicklyoutpaces that of the smaller fraction) and as a function ofsequestration of amplification reagents and resources (as the largerfraction is amplified, it preferentially utilizes primers and otheramplification reagents).

While some of these difficulties may be addressed by utilizing differentsequencing systems, such as single molecule systems that don't requireamplification, the single molecule systems, as well as the ensemblesequencing methods of other next generation sequencing systems, can alsohave requirements for sufficiently large input DNA requirements. Inparticular, single molecule sequencing systems like the PacificBiosciences SMRT Sequencing system can have sample input DNArequirements of from 500 nanograms (ng) to upwards of 10 micrograms (g),which is far larger than what can be derived from individual cells oreven small subpopulations of cells. Likewise, other NGS systems can beoptimized for starting amounts of sample DNA in the sample of fromapproximately 50 ng to about 1 μg.

II. Compartmentalization and Characterization of Cells

Disclosed herein, however, are methods and systems for characterizingnucleic acids from small populations of cells, and in some cases, forcharacterizing nucleic acids from individual cells, especially in thecontext of larger populations of cells. The methods and systems provideadvantages of being able to provide the attribution advantages of thenon-amplified single molecule methods with the high throughput of theother next generation systems, with the additional advantages of beingable to process and sequence extremely low amounts of input nucleicacids derivable from individual cells or small collections of cells.

In particular, the methods described herein compartmentalize theanalysis of individual cells or small populations of cells, includinge.g., nucleic acids from individual cells or small groups of cells, andthen allow that analysis to be attributed back to the individual cell orsmall group of cells from which the nucleic acids were derived. This canbe accomplished regardless of whether the cell population represents a50/50 mix of cell types, a 90/10 mix of cell types, or virtually anyratio of cell types, as well as a complete heterogeneous mix ofdifferent cell types, or any mixture between these. Differing cell typesmay include cells or biologic organisms from different tissue types ofan individual, from different individuals, from differing genera,species, strains, variants, or any combination of any or all of theforegoing. For example, differing cell types may include normal andtumor tissue from an individual, multiple different bacterial species,strains and/or variants from environmental, forensic, microbiome orother samples, or any of a variety of other mixtures of cell types.

In one aspect, the methods and systems described herein, provide for thecompartmentalization, depositing or partitioning of the nucleic acidcontents of individual cells from a sample material containing cells,into discrete compartments or partitions (referred to interchangeablyherein as partitions), where each partition maintains separation of itsown contents from the contents of other partitions. Unique identifiers,e.g., barcodes, may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned cells, in order to allow for the later attribution of thecharacteristics of the individual cells to the particular compartment.

As used herein, in some aspects, the partitions refer to containers orvessels (such as wells, microwells, tubes, through ports in nanoarraysubstrates, e.g., BioTrove nanoarrays, or other containers). In manysome aspects, however, the compartments or partitions comprisepartitions that are flowable within fluid streams. These partitions maybe comprised of, e.g., microcapsules or micro-vesicles that have anouter barrier surrounding an inner fluid center or core, or they may bea porous matrix that is capable of entraining and/or retaining materialswithin its matrix. In some aspects, however, these partitions comprisedroplets of aqueous fluid within a non-aqueous continuous phase, e.g.,an oil phase. A variety of different vessels are described in, forexample, U.S. patent application Ser. No. 13/966,150, filed Aug. 13,2013, the full disclosure of which is incorporated herein by referencein its entirety for all purposes. Likewise, emulsion systems forcreating stable droplets in non-aqueous or oil continuous phases aredescribed in detail in, e.g., U.S. Patent Publication No. 2010/0105112,the full disclosure of which is incorporated herein by reference in itsentirety for all purposes.

In the case of droplets in an emulsion, allocating individual cells todiscrete partitions may generally be accomplished by introducing aflowing stream of cells in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. By providing the aqueous cell-containing stream at acertain concentration level of cells, one can control the level ofoccupancy of the resulting partitions in terms of numbers of cells. Insome cases, where single cell partitions are desired, it may bedesirable to control the relative flow rates of the fluids such that, onaverage, the partitions contain less than one cell per partition, inorder to ensure that those partitions that are occupied, are primarilysingly occupied. Likewise, one may wish to control the flow rate toprovide that a higher percentage of partitions are occupied, e.g.,allowing for only a small percentage of unoccupied partitions. In someaspects, the flows and channel architectures are controlled as to ensurea desired number of singly occupied partitions, less than a certainlevel of unoccupied partitions and less than a certain level of multiplyoccupied partitions.

In many cases, the systems and methods are used to ensure that thesubstantial majority of occupied partitions (partitions containing oneor more microcapsules) include no more than 1 cell per occupiedpartition. In some cases, the partitioning process is controlled suchthat fewer than 25% of the occupied partitions contain more than onecell, and in many cases, fewer than 20% of the occupied partitions havemore than one cell, while in some cases, fewer than 10% or even fewerthan 5% of the occupied partitions include more than one cell perpartition.

Additionally or alternatively, in many cases, it is desirable to avoidthe creation of excessive numbers of empty partitions. While this may beaccomplished by providing sufficient numbers of cells into thepartitioning zone, the poissonian distribution would expectedly increasethe number of partitions that would include multiple cells. As such, inaccordance with aspects described herein, the flow of one or more of thecells, or other fluids directed into the partitioning zone arecontrolled such that, in many cases, no more than 50% of the generatedpartitions are unoccupied, i.e., including less than 1 cell, no morethan 25% of the generated partitions, no more than 10% of the generatedpartitions, may be unoccupied. Further, in some aspects, these flows arecontrolled so as to present non-poissonian distribution of singleoccupied partitions while providing lower levels of unoccupiedpartitions. Restated, in some aspects, the above noted ranges ofunoccupied partitions can be achieved while still providing any of thesingle occupancy rates described above. For example, in many cases, theuse of the systems and methods described herein creates resultingpartitions that have multiple occupancy rates of from less than 25%,less than 20%, less than 15%, less than 10%, and in many cases, lessthan 5%, while having unoccupied partitions of from less than 50%, lessthan 40%, less than 30%, less than 20%, less than 10%, and in somecases, less than 5%.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both cells and beads carrying thebarcode oligonucleotides. In particular, in some aspects, a substantialpercentage of the overall occupied partitions will include both a beadand a cell. In particular, it may be desirable to provide that at least50% of the partitions are occupied by at least one cell and at least onebead, or at least 75% of the partitions may be so occupied, or even atleast 80% or at least 90% of the partitions may be so occupied. Further,in those cases where it is desired to provide a single cell and a singlebead within a partition, at least 50% of the partitions can be sooccupied, at least 60%, at least 70%, at least 80% or even at least 90%of the partitions can be so occupied.

Although described in terms of providing substantially singly occupiedpartitions, above, in certain cases, it is desirable to provide multiplyoccupied partitions, e.g., containing two, three, four or more cellsand/or beads within a single partition. Accordingly, as noted above, theflow characteristics of the cell and/or bead containing fluids andpartitioning fluids may be controlled to provide for such multiplyoccupied partitions. In particular, the flow parameters may becontrolled to provide a desired occupancy rate at greater than 50% ofthe partitions, greater than 75%, and in some cases greater than 80%,90%, 95%, or higher.

Additionally, in many cases, the multiple beads within a singlepartition may comprise different reagents associated therewith. In suchcases, it may be advantageous to introduce different beads into a commonchannel or droplet generation junction, from different bead sources,i.e., containing different associated reagents, through differentchannel inlets into such common channel or droplet generation junction.In such cases, the flow and frequency of the different beads into thechannel or junction may be controlled to provide for the desired ratioof microcapsules from each source, while ensuring the desired pairing orcombination of such beads into a partition with the desired number ofcells.

The partitions described herein are often characterized by havingextremely small volumes, e.g., less than 10 μL, less than 5 μL, lessthan 1 μL, less than 900 picoliters (pL), less than 800 pL, less than700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than300 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than20 pL, less than 10 pL, less than 1 pL, less than 500 nanoliters (nL),or even less than 100 nL, 50 nL, or even less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than 1000 pL, less than 900 pL, lessthan 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, lessthan 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, lessthan 50 pL, less than 20 pL, less than 10 pL, or even less than 1 pL.Where co-partitioned with beads, it will be appreciated that the samplefluid volume, e.g., including co-partitioned cells, within thepartitions may be less than 90% of the above described volumes, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, or even less than 10% the above describedvolumes.

As is described elsewhere herein, partitioning species may generate apopulation of partitions. In such cases, any suitable number ofpartitions can be generated to generate the population of partitions.For example, in a method described herein, a population of partitionsmay be generated that comprises at least about 1,000 partitions, atleast about 5,000 partitions, at least about 10,000 partitions, at leastabout 50,000 partitions, at least about 100,000 partitions, at leastabout 500,000 partitions, at least about 1,000,000 partitions, at leastabout 5,000,000 partitions at least about 10,000,000 partitions, atleast about 50,000,000 partitions, at least about 100,000,000partitions, at least about 500,000,000 partitions or at least about1,000,000,000 partitions. Moreover, the population of partitions maycomprise both unoccupied partitions (e.g., empty partitions) andoccupied partitions

In certain cases, microfluidic channel networks are particularly suitedfor generating partitions as described herein. Examples of suchmicrofluidic devices include those described in detail in ProvisionalU.S. Patent Application No. 61/977,804, filed Apr. 4, 2014, the fulldisclosure of which is incorporated herein by reference in its entiretyfor all purposes. Alternative mechanisms may also be employed in thepartitioning of individual cells, including porous membranes throughwhich aqueous mixtures of cells are extruded into non-aqueous fluids.Such systems are generally available from, e.g., Nanomi, Inc.

An example of a simplified microfluidic channel structure forpartitioning individual cells is illustrated in FIG. 1. As describedelsewhere herein, in some cases, the majority of occupied partitionsinclude no more than one cell per occupied partition and, in some cases,some of the generated partitions are unoccupied. In some cases, though,some of the occupied partitions may include more than one cell. In somecases, the partitioning process may be controlled such that fewer than25% of the occupied partitions contain more than one cell, and in manycases, fewer than 20% of the occupied partitions have more than onecell, while in some cases, fewer than 10% or even fewer than 5% of theoccupied partitions include more than one cell per partition. As shown,the channel structure can include channel segments 102, 104, 106 and 108communicating at a channel junction 110. In operation, a first aqueousfluid 112 that includes suspended cells 114, may be transported alongchannel segment 102 into junction 110, while a second fluid 116 that isimmiscible with the aqueous fluid 112 is delivered to the junction 110from channel segments 104 and 106 to create discrete droplets 118 of theaqueous fluid including individual cells 114, flowing into channelsegment 108.

In some aspects, this second fluid 116 comprises an oil, such as afluorinated oil, that includes a fluorosurfactant for stabilizing theresulting droplets, e.g., inhibiting subsequent coalescence of theresulting droplets. Examples of particularly useful partitioning fluidsand fluorosurfactants are described for example, in U.S. PatentPublication No. 2010/0105112, the full disclosure of which is herebyincorporated herein by reference in its entirety for all purposes.

In other aspects, in addition to or as an alternative to droplet basedpartitioning, cells may be encapsulated within a microcapsule thatcomprises an outer shell or layer or porous matrix in which is entrainedone or more individual cells or small groups of cells, and may includeother reagents. Encapsulation of cells may be carried out by a varietyof processes. In general, such processes combine an aqueous fluidcontaining the cells to be analyzed with a polymeric precursor materialthat may be capable of being formed into a gel or other solid orsemi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli include, e.g., thermal stimuli (eitherheating or cooling), photo-stimuli (e.g., through photo-curing),chemical stimuli (e.g., through crosslinking, polymerization initiationof the precursor (e.g., through added initiators), or the like.

Preparation of microcapsules comprising cells may be carried out by avariety of methods. For example, air knife droplet or aerosol generatorsmay be used to dispense droplets of precursor fluids into gellingsolutions in order to form microcapsules that include individual cellsor small groups of cells. Likewise, membrane based encapsulationsystems, such as those available from, e.g., Nanomi, Inc., may be usedto generate microcapsules as described herein. In some aspects,microfluidic systems like that shown in FIG. 1 may be readily used inencapsulating cells as described herein. In particular, and withreference to FIG. 1, the aqueous fluid comprising the cells and thepolymer precursor material is flowed into channel junction 110, where itis partitioned into droplets 118 comprising the individual cells 114,through the flow of non-aqueous fluid 116. In the case of encapsulationmethods, non-aqueous fluid 116 may also include an initiator to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained cells. Examples of particularlyuseful polymer precursor/initiator pairs include those described in,e.g., U.S. Patent Application Nos. 61/940,318, filed Feb. 7, 2014,61/991,018, Filed May 9, 2014, and U.S. patent application Ser. No.14/316,383, filed Jun. 26, 2014, the full disclosures of which arehereby incorporated herein by reference in their entireties for allpurposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, e.g., a linear polyacrylamide, PEG, or otherlinear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams in channel segments 104 and 106, which initiates thecopolymerization of the acrylamide and BAC into a cross-linked polymernetwork or, hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110 in the formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous first fluid 112 comprisingthe linear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets, resulting in the formation of thegel, e.g., hydrogel, microcapsules 118, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions, e.g., Ca2+, can beused as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling, e.g., upon cooling, or the like. As will beappreciated, in some cases, encapsulated cells can be selectivelyreleasable from the microcapsule, e.g., through passage of time, or uponapplication of a particular stimulus, that degrades the microcapsulesufficiently to allow the cell, or its contents to be released from themicrocapsule, e.g., into an additional partition, such as a droplet. Forexample, in the case of the polyacrylamide polymer described above,degradation of the microcapsule may be accomplished through theintroduction of an appropriate reducing agent, such as DTT or the like,to cleave disulfide bonds that cross link the polymer matrix (See, e.g.,U.S. Provisional Patent Application Nos. 61/940,318, filed Feb. 7, 2014,61/991,018, Filed May 9, 2014, and U.S. patent application Ser. No.14/316,383, filed Jun. 26, 2014, the full disclosures of which arehereby incorporated herein by reference in their entirety for allpurposes.

As will be appreciated, encapsulated cells or cell populations providecertain potential advantages of being storable, and more portable thandroplet based partitioned cells. Furthermore, in some cases, it may bedesirable to allow cells to be analyzed to incubate for a select periodof time, in order to characterize changes in such cells over time,either in the presence or absence of different stimuli. In such cases,encapsulation of individual cells may allow for longer incubation thansimple partitioning in emulsion droplets, although in some cases,droplet partitioned cells may also be incubated form different periodsof time, e.g., at least 10 seconds, at least 30 seconds, at least 1minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, atleast 1 hour, at least 2 hours, at least 5 hours, or at least 10 hoursor more. As alluded to above, the encapsulation of cells may constitutethe partitioning of the cells into which other reagents areco-partitioned. Alternatively, encapsulated cells may be readilydeposited into other partitions, e.g., droplets, as described above.

In accordance with certain aspects, the cells may be partitioned alongwith lysis reagents in order to release the contents of the cells withinthe partition. In such cases, the lysis agents can be contacted with thecell suspension concurrently with, or immediately prior to theintroduction of the cells into the partitioning junction/dropletgeneration zone, e.g., through an additional channel or channelsupstream of channel junction 110. Examples of lysis agents includebioactive reagents, such as lysis enzymes that are used for lysis ofdifferent cell types, e.g., gram positive or negative bacteria, plants,yeast, mammalian, etc., such as lysozymes, achromopeptidase,lysostaphin, labiase, kitalase, lyticase, and a variety of other lysisenzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, Mo.), aswell as other commercially available lysis enzymes. Other lysis agentsmay additionally or alternatively be co-partitioned with the cells tocause the release of the cell's contents into the partitions. Forexample, in some cases, surfactant based lysis solutions may be used tolyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Similarly, lysis methods that employ othermethods may be used, such as electroporation, thermal, acoustic ormechanical cellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of cells that maybe in addition to or in place of droplet partitioning, where any poresize of the encapsulate is sufficiently small to retain nucleic acidfragments of a desired size, following cellular disruption.

In addition to the lysis agents co-partitioned with the cells describedabove, other reagents can also be co-partitioned with the cells,including, for example, DNase and RNase inactivating agents orinhibitors, such as proteinase K, chelating agents, such as EDTA, andother reagents employed in removing or otherwise reducing negativeactivity or impact of different cell lysate components on subsequentprocessing of nucleic acids. In addition, in the case of encapsulatedcells, the cells may be exposed to an appropriate stimulus to releasethe cells or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated cell to allow for the degradation of themicrocapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of oligonucleotides from theirrespective bead or partition. In alternative aspects, this may be adifferent and non-overlapping stimulus, in order to allow anencapsulated cell to be released into a partition at a different timefrom the release of oligonucleotides into the same partition.

Additional reagents may also be co-partitioned with the cells, such asendonucleases to fragment the cell's DNA, DNA polymerase enzymes anddNTPs used to amplify the cell's nucleic acid fragments and to attachthe barcode oligonucleotides to the amplified fragments. Additionalreagents may also include reverse transcriptase enzymes, includingenzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In one example of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., polyC, to the cDNA that are not encoded by thetemplate, such, as at an end of the cDNA. Switch oligos can includesequences complementary to the additional nucleotides, e.g. polyG. Theadditional nucleotides (e.g., polyC) on the cDNA can hybridize to thesequences complementary to the additional nucleotides (e.g., polyG) onthe switch oligo, whereby the switch oligo can be used by the reversetranscriptase as template to further extend the cDNA. Switch oligos maycomprise deoxyribonucleic acids, ribonucleic acids, modified nucleicacids including locked nucleic acids (LNA), or any combination.

In some cases, the length of a switch oligo may be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 nucleotidesor longer.

In some cases, the length of a switch oligo may be at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides or longer.

In some cases, the length of a switch oligo may be at most 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides.

Once the contents of the cells are released into their respectivepartitions, the nucleic acids contained therein may be further processedwithin the partitions. In accordance with the methods and systemsdescribed herein, the nucleic acid contents of individual cells aregenerally provided with unique identifiers such that, uponcharacterization of those nucleic acids they may be attributed as havingbeen derived from the same cell or cells. The ability to attributecharacteristics to individual cells or groups of cells is provided bythe assignment of unique identifiers specifically to an individual cellor groups of cells, which is another advantageous aspect of the methodsand systems described herein. In particular, unique identifiers, e.g.,in the form of nucleic acid barcodes are assigned or associated withindividual cells or populations of cells, in order to tag or label thecell's components (and as a result, its characteristics) with the uniqueidentifiers. These unique identifiers are then used to attribute thecell's components and characteristics to an individual cell or group ofcells. In some aspects, this is carried out by co-partitioning theindividual cells or groups of cells with the unique identifiers. In someaspects, the unique identifiers are provided in the form ofoligonucleotides that comprise nucleic acid barcode sequences that maybe attached to or otherwise associated with the nucleic acid contents ofindividual cells, or to other components of the cells, and particularlyto fragments of those nucleic acids. The oligonucleotides arepartitioned such that as between oligonucleotides in a given partition,the nucleic acid barcode sequences contained therein are the same, butas between different partitions, the oligonucleotides can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from 6 to about 20 ormore nucleotides within the sequence of the oligonucleotides. In somecases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides maybe completely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned oligonucleotides can also comprise other functionalsequences useful in the processing of the nucleic acids from theco-partitioned cells. These sequences include, e.g., targeted orrandom/universal amplification primer sequences for amplifying thegenomic DNA from the individual cells within the partitions whileattaching the associated barcode sequences, sequencing primers or primerrecognition sites, hybridization or probing sequences, e.g., foridentification of presence of the sequences or for pulling down barcodednucleic acids, or any of a number of other potential functionalsequences. Again, co-partitioning of oligonucleotides and associatedbarcodes and other functional sequences, along with sample materials isdescribed in, for example, U.S. Patent Application Nos. 61/940,318,filed Feb. 7, 2014, 61/991,018, filed May 9, 2014, and U.S. patentapplication Ser. No. 14/316,383, filed Jun. 26, 2014, as well as U.S.patent application Ser. No. 14/175,935, filed Feb. 7, 2014, the fulldisclosures of which are incorporated herein by reference in theirentireties for all purposes. As will be appreciated other mechanisms ofco-partitioning oligonucleotides may also be employed, including, e.g.,coalescence of two or more droplets, where one droplet containsoligonucleotides, or microdispensing of oligonucleotides intopartitions, e.g., droplets within microfluidic systems.

Briefly, in one example, beads, microparticles or microcapsules areprovided that each include large numbers of the above describedoligonucleotides releasably attached to the beads, where all of theoligonucleotides attached to a particular bead will include the samenucleic acid barcode sequence, but where a large number of diversebarcode sequences are represented across the population of beads used.In particularly useful examples, hydrogel beads, e.g., comprisingpolyacrylamide polymer matrices, are used as a solid support anddelivery vehicle for the oligonucleotides into the partitions, as theyare capable of carrying large numbers of oligonucleotide molecules, andmay be configured to release those oligonucleotides upon exposure to aparticular stimulus, as described elsewhere herein. In some cases, thepopulation of beads will provide a diverse barcode sequence library thatincludes at least 1,000 different barcode sequences, at least 5,000different barcode sequences, at least 10,000 different barcodesequences, at least at least 50,000 different barcode sequences, atleast 100,000 different barcode sequences, at least 1,000,000 differentbarcode sequences, at least 5,000,000 different barcode sequences, or atleast 10,000,000 different barcode sequences. Additionally, each beadcan be provided with large numbers of oligonucleotide moleculesattached. In particular, the number of molecules of oligonucleotidesincluding the barcode sequence on an individual bead can be at least1,000 oligonucleotide molecules, at least 5,000 oligonucleotidemolecules, at least 10,000 oligonucleotide molecules, at least 50,000oligonucleotide molecules, at least 100,000 oligonucleotide molecules,at least 500,000 oligonucleotides, at least 1,000,000 oligonucleotidemolecules, at least 5,000,000 oligonucleotide molecules, at least10,000,000 oligonucleotide molecules, at least 50,000,000oligonucleotide molecules, at least 100,000,000 oligonucleotidemolecules, and in some cases at least 1 billion oligonucleotidemolecules.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least 1,000 different barcode sequences, at least 5,000different barcode sequences, at least 10,000 different barcodesequences, at least at least 50,000 different barcode sequences, atleast 100,000 different barcode sequences, at least 1,000,000 differentbarcode sequences, at least 5,000,000 different barcode sequences, or atleast 10,000,000 different barcode sequences. Additionally, eachpartition of the population can include at least 1,000 oligonucleotidemolecules, at least 5,000 oligonucleotide molecules, at least 10,000oligonucleotide molecules, at least 50,000 oligonucleotide molecules, atleast 100,000 oligonucleotide molecules, at least 500,000oligonucleotides, at least 1,000,000 oligonucleotide molecules, at least5,000,000 oligonucleotide molecules, at least 10,000,000 oligonucleotidemolecules, at least 50,000,000 oligonucleotide molecules, at least100,000,000 oligonucleotide molecules, and in some cases at least 1billion oligonucleotide molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known barcode sequences set may provide greater assurance ofidentification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The oligonucleotides are releasable from the beads upon the applicationof a particular stimulus to the beads. In some cases, the stimulus maybe a photo-stimulus, e.g., through cleavage of a photo-labile linkagethat releases the oligonucleotides. In other cases, a thermal stimulusmay be used, where elevation of the temperature of the beads environmentwill result in cleavage of a linkage or other release of theoligonucleotides form the beads. In still other cases, a chemicalstimulus is used that cleaves a linkage of the oligonucleotides to thebeads, or otherwise results in release of the oligonucleotides from thebeads. Examples of this type of system are described in U.S. patentapplication Ser. No. 13/966,150, filed Aug. 13, 2013, as well as U.S.Provisional Patent Application Nos. 61/940,318, filed Feb. 7, 2014,61/991,018, Filed May 9, 2014, and U.S. patent application Ser. No.14/316,383, filed Jun. 26, 2014, the full disclosures of which arehereby incorporated herein by reference n their entireties for allpurposes. In one case, such compositions include the polyacrylamidematrices described above for encapsulation of cells, and may be degradedfor release of the attached oligonucleotides through exposure to areducing agent, such as DTT.

In accordance with the methods and systems described herein, the beadsincluding the attached oligonucleotides are co-partitioned with theindividual cells, such that a single bead and a single cell arecontained within an individual partition. As noted above, while singlecell/single bead occupancy is the most desired state, it will beappreciated that multiply occupied partitions (either in terms of cells,beads or both), or unoccupied partitions (either in terms of cells,beads or both) will often be present. An example of a microfluidicchannel structure for co-partitioning cells and beads comprising barcodeoligonucleotides is schematically illustrated in FIG. 2. As describedelsewhere herein, in some aspects, a substantial percentage of theoverall occupied partitions will include both a bead and a cell and, insome cases, some of the partitions that are generated will beunoccupied. In some cases, some of the partitions may have beads andcells that are not partitioned 1:1. In some cases, it may be desirableto provide multiply occupied partitions, e.g., containing two, three,four or more cells and/or beads within a single partition. As shown,channel segments 202, 204, 206, 208 and 210 are provided in fluidcommunication at channel junction 212. An aqueous stream comprising theindividual cells 214, is flowed through channel segment 202 towardchannel junction 212. As described above, these cells may be suspendedwithin an aqueous fluid, or may have been pre-encapsulated, prior to thepartitioning process.

Concurrently, an aqueous stream comprising the barcode carrying beads216, is flowed through channel segment 204 toward channel junction 212.A non-aqueous partitioning fluid 216 is introduced into channel junction212 from each of side channels 206 and 208, and the combined streams areflowed into outlet channel 210. Within channel junction 212, the twocombined aqueous streams from channel segments 202 and 204 are combined,and partitioned into droplets 218, that include co-partitioned cells 214and beads 216. As noted previously, by controlling the flowcharacteristics of each of the fluids combining at channel junction 212,as well as controlling the geometry of the channel junction, one canoptimize the combination and partitioning to achieve a desired occupancylevel of beads, cells or both, within the partitions 218 that aregenerated.

In some cases, lysis agents, e.g., cell lysis enzymes, may be introducedinto the partition with the bead stream, e.g., flowing through channelsegment 204, such that lysis of the cell only commences at or after thetime of partitioning. Additional reagents may also be added to thepartition in this configuration, such as endonucleases to fragment thecell's DNA, DNA polymerase enzyme and dNTPs used to amplify the cell'snucleic acid fragments and to attach the barcode oligonucleotides to theamplified fragments. As noted above, in many cases, a chemical stimulus,such as DTT, may be used to release the barcodes from their respectivebeads into the partition. In such cases, it may be particularlydesirable to provide the chemical stimulus along with thecell-containing stream in channel segment 202, such that release of thebarcodes only occurs after the two streams have been combined, e.g.,within the partitions 218. Where the cells are encapsulated, however,introduction of a common chemical stimulus, e.g., that both releases theoligonucleotides form their beads, and releases cells from theirmicrocapsules may generally be provided from a separate additional sidechannel (not shown) upstream of or connected to channel junction 212.

As will be appreciated, a number of other reagents may be co-partitionedalong with the cells, beads, lysis agents and chemical stimuli,including, for example, protective reagents, like proteinase K,chelators, nucleic acid extension, replication, transcription oramplification reagents such as polymerases, reverse transcriptases,transposases which can be used for transposon based methods (e.g.,Nextera), nucleoside triphosphates or NTP analogues, primer sequencesand additional cofactors such as divalent metal ions used in suchreactions, ligation reaction reagents, such as ligase enzymes andligation sequences, dyes, labels, or other tagging reagents.

The channel networks, e.g., as described herein, can be fluidly coupledto appropriate fluidic components. For example, the inlet channelsegments, e.g., channel segments 202, 204, 206 and 208 are fluidlycoupled to appropriate sources of the materials they are to deliver tochannel junction 212. For example, channel segment 202 will be fluidlycoupled to a source of an aqueous suspension of cells 214 to beanalyzed, while channel segment 204 would be fluidly coupled to a sourceof an aqueous suspension of beads 216. Channel segments 206 and 208would then be fluidly connected to one or more sources of thenon-aqueous fluid. These sources may include any of a variety ofdifferent fluidic components, from simple reservoirs defined in orconnected to a body structure of a microfluidic device, to fluidconduits that deliver fluids from off-device sources, manifolds, or thelike. Likewise, the outlet channel segment 210 may be fluidly coupled toa receiving vessel or conduit for the partitioned cells. Again, this maybe a reservoir defined in the body of a microfluidic device, or it maybe a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

FIG. 8 shows images of individual Jurkat cells co-partitioned along withbarcode oligonucleotide containing beads in aqueous droplets in anaqueous in oil emulsion. As illustrated, individual cells may be readilyco-partitioned with individual beads. As will be appreciated,optimization of individual cell loading may be carried out by a numberof methods, including by providing dilutions of cell populations intothe microfluidic system in order to achieve the desired cell loading perpartition as described elsewhere herein.

In operation, once lysed, the nucleic acid contents of the individualcells are then available for further processing within the partitions,including, e.g., fragmentation, amplification and barcoding, as well asattachment of other functional sequences. As noted above, fragmentationmay be accomplished through the co-partitioning of shearing enzymes,such as endonucleases, in order to fragment the nucleic acids intosmaller fragments. These endonucleases may include restrictionendonucleases, including type II and type IIs restriction endonucleasesas well as other nucleic acid cleaving enzymes, such as nickingendonucleases, and the like. In some cases, fragmentation may not bedesired, and full length nucleic acids may be retained within thepartitions, or in the case of encapsulated cells or cell contents,fragmentation may be carried out prior to partitioning, e.g., throughenzymatic methods, e.g., those described herein, or through mechanicalmethods, e.g., mechanical, acoustic or other shearing.

Once co-partitioned, and the cells are lysed to release their nucleicacids, the oligonucleotides disposed upon the bead may be used tobarcode and amplify fragments of those nucleic acids. A particularlyelegant process for use of these barcode oligonucleotides in amplifyingand barcoding fragments of sample nucleic acids is described in detailin U.S. Provisional Patent Application Nos. 61/940,318, filed Feb. 7,2014, 61/991,018, Filed May 9, 2014, and U.S. patent application Ser.No. 14/316,383, filed Jun. 26, 2014, and previously incorporated byreference. Briefly, in one aspect, the oligonucleotides present on thebeads that are co-partitioned with the cells, are released from theirbeads into the partition with the cell's nucleic acids. Theoligonucleotides can include, along with the barcode sequence, a primersequence at its 5′end. This primer sequence may be a randomoligonucleotide sequence intended to randomly prime numerous differentregions on the cell's nucleic acids, or it may be a specific primersequence targeted to prime upstream of a specific targeted region of thecell's genome.

Once released, the primer portion of the oligonucleotide can anneal to acomplementary region of the cell's nucleic acid. Extension reactionreagents, e.g., DNA polymerase, nucleoside triphosphates, co-factors(e.g., Mg2+ or Mn2+), that are also co-partitioned with the cells andbeads, then extend the primer sequence using the cell's nucleic acid asa template, to produce a complementary fragment to the strand of thecell's nucleic acid to which the primer annealed, which complementaryfragment includes the oligonucleotide and its associated barcodesequence. Annealing and extension of multiple primers to differentportions of the cell's nucleic acids will result in a large pool ofoverlapping complementary fragments of the nucleic acid, each possessingits own barcode sequence indicative of the partition in which it wascreated. In some cases, these complementary fragments may themselves beused as a template primed by the oligonucleotides present in thepartition to produce a complement of the complement that again, includesthe barcode sequence. In some cases, this replication process isconfigured such that when the first complement is duplicated, itproduces two complementary sequences at or near its termini, to allowformation of a hairpin structure or partial hairpin structure, thereduces the ability of the molecule to be the basis for producingfurther iterative copies. As described herein, the cell's nucleic acidsmay include any desired nucleic acids within the cell including, forexample, the cell's DNA, e.g., genomic DNA, RNA, e.g., messenger RNA,and the like. For example, in some cases, the methods and systemsdescribed herein are used in characterizing expressed mRNA, including,e.g., the presence and quantification of such mRNA, and may include RNAsequencing processes as the characterization process. Alternatively oradditionally, the reagents partitioned along with the cells may includereagents for the conversion of mRNA into cDNA, e.g., reversetranscriptase enzymes and reagents, to facilitate sequencing processeswhere DNA sequencing is employed. In some cases, where the nucleic acidsto be characterized comprise RNA, e.g., mRNA, schematic illustration ofone example of this is shown in FIG. 3.

As shown, oligonucleotides that include a barcode sequence areco-partitioned in, e.g., a droplet 302 in an emulsion, along with asample nucleic acid 304. As noted elsewhere herein, the oligonucleotides308 may be provided on a bead 306 that is co-partitioned with the samplenucleic acid 304, which oligonucleotides are releasable from the bead306, as shown in panel A. The oligonucleotides 308 include a barcodesequence 312, in addition to one or more functional sequences, e.g.,sequences 310, 314 and 316. For example, oligonucleotide 308 is shown ascomprising barcode sequence 312, as well as sequence 310 that mayfunction as an attachment or immobilization sequence for a givensequencing system, e.g., a P5 sequence used for attachment in flow cellsof an Illumina Hiseq® or Miseq® system. As shown, the oligonucleotidesalso include a primer sequence 316, which may include a random ortargeted N-mer for priming replication of portions of the sample nucleicacid 304. Also included within oligonucleotide 308 is a sequence 314which may provide a sequencing priming region, such as a “read1” or R1priming region, that is used to prime polymerase mediated, templatedirected sequencing by synthesis reactions in sequencing systems. Aswill be appreciated, the functional sequences may be selected to becompatible with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof. In many cases, the barcode sequence 312,immobilization sequence 310 and R1 sequence 314 may be common to all ofthe oligonucleotides attached to a given bead. The primer sequence 316may vary for random N-mer primers, or may be common to theoligonucleotides on a given bead for certain targeted applications.

As will be appreciated, in some cases, the functional sequences mayinclude primer sequences useful for RNA-seq applications. For example,in some cases, the oligonucleotides may include poly-T primers forpriming reverse transcription of RNA for RNA-seq. In still other cases,oligonucleotides in a given partition, e.g., included on an individualbead, may include multiple types of primer sequences in addition to thecommon barcode sequences, such as both DNA-sequencing and RNA sequencingprimers, e.g., poly-T primer sequences included within theoligonucleotides coupled to the bead. In such cases, a singlepartitioned cell may be both subjected to DNA and RNA sequencingprocesses.

Based upon the presence of primer sequence 316, the oligonucleotides canprime the sample nucleic acid as shown in panel B, which allows forextension of the oligonucleotides 308 and 308 a using polymerase enzymesand other extension reagents also co-partitioned with the bead 306 andsample nucleic acid 304. As shown in panel C, following extension of theoligonucleotides that, for random N-mer primers, would anneal tomultiple different regions of the sample nucleic acid 304; multipleoverlapping complements or fragments of the nucleic acid are created,e.g., fragments 318 and 320. Although including sequence portions thatare complementary to portions of sample nucleic acid, e.g., sequences322 and 324, these constructs are generally referred to herein ascomprising fragments of the sample nucleic acid 304, having the attachedbarcode sequences.

The barcoded nucleic acid fragments may then be subjected tocharacterization, e.g., through sequence analysis, or they may befurther amplified in the process, as shown in panel D. For example,additional oligonucleotides, e.g., oligonucleotide 308 b, also releasedfrom bead 306, may prime the fragments 318 and 320. This shown in forfragment 318. In particular, again, based upon the presence of therandom N-mer primer 316 b in oligonucleotide 308 b (which in many casescan be different from other random N-mers in a given partition, e.g.,primer sequence 316), the oligonucleotide anneals with the fragment 318,and is extended to create a complement 326 to at least a portion offragment 318 which includes sequence 328, that comprises a duplicate ofa portion of the sample nucleic acid sequence. Extension of theoligonucleotide 308 b continues until it has replicated through theoligonucleotide portion 308 of fragment 318. As noted elsewhere herein,and as illustrated in panel D, the oligonucleotides may be configured toprompt a stop in the replication by the polymerase at a desired point,e.g., after replicating through sequences 316 and 314 of oligonucleotide308 that is included within fragment 318. As described herein, this maybe accomplished by different methods, including, for example, theincorporation of different nucleotides and/or nucleotide analogues thatare not capable of being processed by the polymerase enzyme used. Forexample, this may include the inclusion of uracil containing nucleotideswithin the sequence region 312 to prevent a non-uracil tolerantpolymerase to cease replication of that region. As a result a fragment326 is created that includes the full-length oligonucleotide 308 b atone end, including the barcode sequence 312, the attachment sequence310, the R1 primer region 314, and the random N-mer sequence 316 b. Atthe other end of the sequence may be included the complement 316′ to therandom N-mer of the first oligonucleotide 308, as well as a complementto all or a portion of the R1 sequence, shown as sequence 314′. The R1sequence 314 and its complement 314′ are then able to hybridize togetherto form a partial hairpin structure 328. As will be appreciated becausethe random N-mers differ among different oligonucleotides, thesesequences and their complements would not be expected to participate inhairpin formation, e.g., sequence 316′, which is the complement torandom N-mer 316, would not be expected to be complementary to randomN-mer sequence 316 b. This would not be the case for other applications,e.g., targeted primers, where the N-mers would be common amongoligonucleotides within a given partition.

By forming these partial hairpin structures, it allows for the removalof first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 326.

In general, the amplification of the cell's nucleic acids is carried outuntil the barcoded overlapping fragments within the partition constituteat least 1× coverage of the particular portion or all of the cell'sgenome, at least 2×, at least 3×, at least 4×, at least 5×, at least10×, at least 20×, at least 40× or more coverage of the genome or itsrelevant portion of interest. Once the barcoded fragments are produced,they may be directly sequenced on an appropriate sequencing system,e.g., an Illumina Hiseq®, Miseq® or X10 system, or they may be subjectedto additional processing, such as further amplification, attachment ofother functional sequences, e.g., second sequencing primers, for reversereads, sample index sequences, and the like.

All of the fragments from multiple different partitions may then bepooled for sequencing on high throughput sequencers as described herein,where the pooled fragments comprise a large number of fragments derivedfrom the nucleic acids of different cells or small cell populations, butwhere the fragments from the nucleic acids of a given cell will sharethe same barcode sequence. In particular, because each fragment is codedas to its partition of origin, and consequently its single cell or smallpopulation of cells, the sequence of that fragment may be attributedback to that cell or those cells based upon the presence of the barcode,which will also aid in applying the various sequence fragments frommultiple partitions to assembly of individual genomes for differentcells. This is schematically illustrated in FIG. 4. As shown in oneexample, a first nucleic acid 404 from a first cell 400, and a secondnucleic acid 406 from a second cell 402 are each partitioned along withtheir own sets of barcode oligonucleotides as described above. Thenucleic acids may comprise a chromosome, entire genome or other largenucleic acid from the cells.

Within each partition, each cell's nucleic acids 404 and 406 is thenprocessed to separately provide overlapping set of second fragments ofthe first fragment(s), e.g., second fragment sets 408 and 410. Thisprocessing also provides the second fragments with a barcode sequencethat is the same for each of the second fragments derived from aparticular first fragment. As shown, the barcode sequence for secondfragment set 408 is denoted by “1” while the barcode sequence forfragment set 410 is denoted by “2”. A diverse library of barcodes may beused to differentially barcode large numbers of different fragment sets.However, it is not necessary for every second fragment set from adifferent first fragment to be barcoded with different barcodesequences. In fact, in many cases, multiple different first fragmentsmay be processed concurrently to include the same barcode sequence.Diverse barcode libraries are described in detail elsewhere herein.

The barcoded fragments, e.g., from fragment sets 408 and 410, may thenbe pooled for sequencing using, for example, sequence by synthesistechnologies available from Illumina or Ion Torrent division ofThermo-Fisher, Inc. Once sequenced, the sequence reads 412 can beattributed to their respective fragment set, e.g., as shown inaggregated reads 414 and 416, at least in part based upon the includedbarcodes, and in some cases, in part based upon the sequence of thefragment itself. The attributed sequence reads for each fragment set arethen assembled to provide the assembled sequence for each cell's nucleicacids, e.g., sequences 418 and 420, which in turn, may be attributed toindividual cells, e.g., cells 400 and 402.

While described in terms of analyzing the genetic material presentwithin cells, the methods and systems described herein may have muchbroader applicability, including the ability to characterize otheraspects of individual cells or cell populations, by allowing for theallocation of reagents to individual cells, and providing for theattributable analysis or characterization of those cells in response tothose reagents. These methods and systems are particularly valuable inbeing able to characterize cells for, e.g., research, diagnostic,pathogen identification, and many other purposes. By way of example, awide range of different cell surface features, e.g., cell surfaceproteins like cluster of differentiation or CD proteins, havesignificant diagnostic relevance in characterization of diseases likecancer.

In one particularly useful application, the methods and systemsdescribed herein may be used to characterize cell features, such as cellsurface features, e.g., proteins, receptors, etc. In particular, themethods described herein may be used to attach reporter molecules tothese cell features, that when partitioned as described above, may bebarcoded and analyzed, e.g., using DNA sequencing technologies, toascertain the presence, and in some cases, relative abundance orquantity of such cell features within an individual cell or populationof cells.

In a particular example, a library of potential cell binding ligands,e.g., antibodies, antibody fragments, cell surface receptor bindingmolecules, or the like, may be provided associated with a first set ofnucleic acid reporter molecules, e.g., where a different reporteroligonucleotide sequence is associated with a specific ligand, andtherefore capable of binding to a specific cell surface feature. In someaspects, different members of the library may be characterized by thepresence of a different oligonucleotide sequence label, e.g., anantibody to a first type of cell surface protein or receptor would haveassociated with it a first known reporter oligonucleotide sequence,while an antibody to a second receptor protein would have a differentknown reporter oligonucleotide sequence associated with it. Prior toco-partitioning, the cells would be incubated with the library ofligands, that may represent antibodies to a broad panel of differentcell surface features, e.g., receptors, proteins, etc., and whichinclude their associated reporter oligonucleotides. Unbound ligands arewashed from the cells, and the cells are then co-partitioned along withthe barcode oligonucleotides described above. As a result, thepartitions will include the cell or cells, as well as the bound ligandsand their known, associated reporter oligonucleotides.

Without the need for lysing the cells within the partitions, one couldthen subject the reporter oligonucleotides to the barcoding operationsdescribed above for cellular nucleic acids, to produce barcoded,reporter oligonucleotides, where the presence of the reporteroligonucleotides can be indicative of the presence of the particularcell surface feature, and the barcode sequence will allow theattribution of the range of different cell surface features to a givenindividual cell or population of cells based upon the barcode sequencethat was co-partitioned with that cell or population of cells. As aresult, one may generate a cell-by-cell profile of the cell surfacefeatures within a broader population of cells. This aspect of themethods and systems described herein, is described in greater detailbelow.

This example is schematically illustrated in FIG. 5. As shown, apopulation of cells, represented by cells 502 and 504 are incubated witha library of cell surface associated reagents, e.g., antibodies, cellsurface binding proteins, ligands or the like, where each different typeof binding group includes an associated nucleic acid reporter moleculeassociated with it, shown as ligands and associated reporter molecules506, 508, 510 and 512 (with the reporter molecules being indicated bythe differently shaded circles). Where the cell expresses the surfacefeatures that are bound by the library, the ligands and their associatedreporter molecules can become associated or coupled with the cellsurface. Individual cells are then partitioned into separate partitions,e.g., droplets 514 and 516, along with their associated ligand/reportermolecules, as well as an individual barcode oligonucleotide bead asdescribed elsewhere herein, e.g., beads 522 and 524, respectively. Aswith other examples described herein, the barcoded oligonucleotides arereleased from the beads and used to attach the barcode sequence thereporter molecules present within each partition with a barcode that iscommon to a given partition, but which varies widely among differentpartitions. For example, as shown in FIG. 5, the reporter molecules thatassociate with cell 502 in partition 514 are barcoded with barcodesequence 518, while the reporter molecules associated with cell 504 inpartition 516 are barcoded with barcode 520. As a result, one isprovided with a library of oligonucleotides that reflects the surfaceligands of the cell, as reflected by the reporter molecule, but which issubstantially attributable to an individual cell by virtue of a commonbarcode sequence, allowing a single cell level profiling of the surfacecharacteristics of the cell. As will be appreciated, this process is notlimited to cell surface receptors but may be used to identify thepresence of a wide variety of specific cell structures, chemistries orother characteristics.

III. Barcoding

Downstream applications, for example DNA sequencing, may rely on thebarcodes to identify the origin of a sequence and, for example, toassemble a larger sequence from sequenced fragments. Therefore, it maybe desirable to add barcodes to the polynucleotide fragments generatedby the methods described herein. Barcodes may be of a variety ofdifferent formats, including polynucleotide barcodes. Depending upon thespecific application, barcodes may be attached to polynucleotidefragments in a reversible or irreversible manner. Barcodes may alsoallow for identification and/or quantification of individualpolynucleotide fragments during sequencing.

Barcodes may be loaded into partitions so that one or more barcodes areintroduced into a particular partition. Each partition may contain adifferent set of barcodes. This may be accomplished by directlydispensing the barcodes into the partitions, enveloping the barcodes(e.g., in a droplet of an emulsion), or by placing the barcodes within acontainer that is placed in a partition (e.g., a microcapsule).

For example, a population of microcapsules may be prepared such that afirst microcapsule in the population comprises multiple copies ofidentical barcodes (e.g., polynucleotide bar codes, etc.) and a secondmicrocapsule in the population comprises multiple copies of a barcodethat differs from the barcode within the first microcapsule. In somecases, the population of microcapsules may comprise multiplemicrocapsules (e.g., greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000, 100000,1000000, 10000000, 100000000, or 1000000000 microcapsules), eachcontaining multiple copies of a barcode that differs from that containedin the other microcapsules. In some cases, the population may comprisegreater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, 10000000,100000000, or 1000000000 microcapsules with identical sets of barcodes.In some cases, the population may comprise greater than about 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000,10000, 100000, 1000000, 10000000, 100000000, or 1000000000microcapsules, wherein the microcapsules each comprise a differentcombination of barcodes. For example, in some cases the differentcombinations overlap, such that a first microcapsule may comprise, e.g.,barcodes A, B, and C, while a second microcapsule may comprise barcodesA, B, and D. In another example, the different combinations do notoverlap, such that a first microcapsule may comprise, e.g., barcodes A,B, and C, while a second microcapsule may comprise barcodes D, E, and F.The use of microcapsules is, of course, optional. All of thecombinations described above, and throughout this disclosure, may alsobe generated by dispending barcodes (and other reagents) directly intopartitions (e.g., microwells).

The barcodes may be loaded into the partitions at an expected orpredicted ratio of barcodes per species to be barcoded (e.g.,polynucleotide fragment, strand of polynucleotide, cell, etc.). In somecases, the barcodes are loaded into partitions such that more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or200000 barcodes are loaded per species. In some cases, the barcodes areloaded in the partitions so that less than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes areloaded per species. In some cases, the average number of barcodes loadedper species is less than, or greater than, about 0.0001, 0.001, 0.01,0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000,or 200000 barcodes per species.

When more than one barcode is present per polynucleotide fragment, suchbarcodes may be copies of the same barcode, or multiple differentbarcodes. For example, the attachment process may be designed to attachmultiple identical barcodes to a single polynucleotide fragment, ormultiple different barcodes to the polynucleotide fragment.

The methods provided herein may comprise loading a partition (e.g., amicrowell, droplet of an emulsion) with the reagents necessary for theattachment of barcodes to polynucleotide fragments. In the case ofligation reactions, reagents including restriction enzymes, ligaseenzymes, buffers, adapters, barcodes and the like may be loaded into apartition. In the case barcoding by amplification, reagents includingprimers, DNA polymerases, DNTPs, buffers, barcodes and the like may beloaded into a partition. As described throughout this disclosure, thesereagents may be loaded directly into the partition, or via a containersuch as a microcapsule. If the reagents are not disposed within acontainer, they may be loaded into a partition (e.g., a microwell) whichmay then be sealed with a wax or oil until the reagents are used.

Barcodes may be ligated to a polynucleotide fragment using sticky orblunt ends. Barcoded polynucleotide fragments may also be generated byamplifying a polynucleotide fragment with primers comprising barcodes.

Barcodes may be assembled combinatorially, from smaller componentsdesigned to assemble in a modular format. For example, three modules,1A, 1B, and 1C may be combinatorially assembled to produce barcode 1ABC.Such combinatorial assembly may significantly reduce the cost ofsynthesizing a plurality of barcodes. For example, a combinatorialsystem consisting of 3 A modules, 3 B modules, and 3 C modules maygenerate 3*3*3=27 possible barcode sequences from only 9 modules.

Barcoding and beads of the present disclosure may be performed and usedas described in, for example, WO2014/028537 and WO 2014/124338, each ofwhich is entirely incorporated herein by reference.

IV. Applications of Single Cell Analysis

There are a wide variety of different applications of the single cellprocessing and analysis methods and systems described herein, includinganalysis of specific individual ells, analysis of different cell typeswithin populations of differing cell types, analysis andcharacterization of large populations of cells for environmental, humanhealth, epidemiological forensic, or any of a wide variety of differentapplications.

A particularly valuable application of the single cell analysisprocesses described herein is in the sequencing and characterization ofcancer cells. In particular, conventional analytical techniques,including the ensemble sequencing processes alluded to above, are nothighly adept at picking small variations in genomic make-up of cancercells, particularly where those exist in a sea of normal tissue cells.Further, even as between tumor cells, wide variations can exist and canbe masked by the ensemble approaches to sequencing (See, e.g., Patel, etal., Single-cell RNA-seq highlights intratumoral heterogeneity inprimary glioblastoma, Science DOI: 10.1126/science. 1254257 (Publishedonline Jun. 12, 2014). Cancer cells may be derived from solid tumors,hematological malignancies, cell lines, or obtained as circulating tumorcells, and subjected to the partitioning processes described above. Uponanalysis, one can identify individual cell sequences as deriving from asingle cell or small group of cells, and distinguish those over normaltissue cell sequences. Further, as described in co-pending U.S.Provisional Patent Application No. 62/017,808, filed Jun. 26, 2014, thefull disclosures of which is hereby incorporated herein by reference inits entirety for all purposes, one may also obtain phased sequenceinformation from each cell, allowing clearer characterization of thehaplotype variants within a cancer cell. The single cell analysisapproach is particularly useful for systems and methods involving lowquantities of input nucleic acids, as described in co-pending U.S.Provisional Patent Application No. 62/017,580, filed Jun. 26, 2014, thefull disclosures of which is hereby incorporated herein by reference inits entirety for all purposes.

As with cancer cell analysis, the analysis and diagnosis of fetal healthor abnormality through the analysis of fetal cells is a difficult taskusing conventional techniques. In particular, in the absence ofrelatively invasive procedures, such as amniocentesis obtaining fetalcell samples can employ harvesting those cells from the maternalcirculation. As will be appreciated, such circulating fetal cells makeup an extremely small fraction of the overall cellular population ofthat circulation. As a result complex analyses are performed in order tocharacterize what of the obtained data is likely derived from fetalcells as opposed to maternal cells. By employing the single cellcharacterization methods and systems described herein, however, one canattribute genetic make up to individual cells, and categorize thosecells as maternal or fetal based upon their respective genetic make-up.Further, the genetic sequence of fetal cells may be used to identify anyof a number of genetic disorders, including, e.g., aneuploidy such asDown syndrome, Edwards syndrome, and Patau syndrome.

The ability to characterize individual cells from larger diversepopulations of cells is also of significant value in both environmentaltesting as well as in forensic analysis, where samples may, by theirnature, be made up of diverse populations of cells and other materialthat “contaminate” the sample, relative to the cells for which thesample is being tested, e.g., environmental indicator organisms, toxicorganisms, and the like for, e.g., environmental and food safetytesting, victim and/or perpetrator cells in forensic analysis for sexualassault, and other violent crimes, and the like.

Additional useful applications of the above described single cellsequencing and characterization processes are in the field ofneuroscience research and diagnosis. In particular, neural cells caninclude long interspersed nuclear elements (LINEs), or ‘jumping’ genesthat can move around the genome, which cause each neuron to differ fromits neighbor cells. Research has shown that the number of LINEs in humanbrain exceeds that of other tissues, e.g., heart and liver tissue, withbetween 80 and 300 unique insertions (See, e.g., Coufal, N. G. et al.Nature 460, 1127-1131 (2009)). These differences have been postulated asbeing related to a person's susceptibility to neuro-logical disorders(see, e.g., Muotri, A. R. et al. Nature 468, 443-446 (2010)), or providethe brain with a diversity with which to respond to challenges. As such,the methods described herein may be used in the sequencing andcharacterization of individual neural cells.

The single cell analysis methods described herein are also useful in theanalysis of gene expression, as noted above, both in terms ofidentification of RNA transcripts and their quantitation. In particular,using the single cell level analysis methods described herein, one canisolate and analyze the RNA transcripts present in individual cells,populations of cells, or subsets of populations of cells. In particular,in some cases, the barcode oligonucleotides may be configured to prime,replicate and consequently yield barcoded fragments of RNA fromindividual cells. For example, in some cases, the barcodeoligonucleotides may include mRNA specific priming sequences, e.g.,poly-T primer segments that allow priming and replication of mRNA in areverse transcription reaction or other targeted priming sequences.Alternatively or additionally, random RNA priming may be carried outusing random N-mer primer segments of the barcode oligonucleotides.

FIG. 6 provides a schematic of one example method for RNA expressionanalysis in individual cells using the methods described herein. Asshown, at operation 602 a cell containing sample is sorted for viablecells, which are quantified and diluted for subsequent partitioning. Atoperation 604, the individual cells separately co-partitioned with gelbeads bearing the barcoding oligonucleotides as described herein. Thecells are lysed and the barcoded oligonucleotides released into thepartitions at operation 606, where they interact with and hybridize tothe mRNA at operation 608, e.g., by virtue of a poly-T primer sequence,which is complementary to the poly-A tail of the mRNA. Using the poly-Tbarcode oligonucleotide as a priming sequence, a reverse transcriptionreaction is carried out at operation 610 to synthesize a cDNA transcriptof the mRNA that includes the barcode sequence. The barcoded cDNAtranscripts are then subjected to additional amplification at operation612, e.g., using a PCR process, purification at operation 614, beforethey are placed on a nucleic acid sequencing system for determination ofthe cDNA sequence and its associated barcode sequence(s). In some cases,as shown, operations 602 through 608 can occur while the reagents remainin their original droplet or partition, while operations 612 through 616can occur in bulk (e.g., outside of the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled in order to complete operations 612through 616. In some cases, barcode oligonucleotides may be digestedwith exonucleases after the emulsion is broken. Exonuclease activity canbe inhibited by ethylenediaminetetraacetic acid (EDTA) following primerdigestion. In some cases, operation 610 may be performed either withinthe partitions based upon co-partitioning of the reverse transcriptionmixture, e.g., reverse transcriptase and associated reagents, or it maybe performed in bulk.

As noted elsewhere herein, the structure of the barcode oligonucleotidesmay include a number of sequence elements in addition to theoligonucleotide barcode sequence. One example of a barcodeoligonucleotide for use in RNA analysis as described above is shown inFIG. 7. As shown, the overall oligonucleotide 702 is coupled to a bead704 by a releasable linkage 706, such as a disulfide linker. Theoligonucleotide may include functional sequences that are used insubsequent processing, such as functional sequence 708, which mayinclude one or more of a sequencer specific flow cell attachmentsequence, e.g., a P5 sequence for Illumina sequencing systems, as wellas sequencing primer sequences, e.g., a R1 primer for Illuminasequencing systems. A barcode sequence 710 is included within thestructure for use in barcoding the sample RNA. An mRNA specific primingsequence, such as poly-T sequence 712 is also included in theoligonucleotide structure. An anchoring sequence segment 714 may beincluded to ensure that the poly-T sequence hybridizes at the sequenceend of the mRNA. This anchoring sequence can include a random shortsequence of nucleotides, e.g., 1-mer, 2-mer, 3-mer or longer sequence,which will ensure that the poly-T segment is more likely to hybridize atthe sequence end of the poly-A tail of the mRNA. An additional sequencesegment 716 may be provided within the oligonucleotide sequence. In somecases, this additional sequence provides a unique molecular sequencesegment, e.g., as a random sequence (e.g., such as a random N-mersequence) that varies across individual oligonucleotides coupled to asingle bead, whereas barcode sequence 710 can be constant amongoligonucleotides tethered to an individual bead. This unique sequenceserves to provide a unique identifier of the starting mRNA molecule thatwas captured, in order to allow quantitation of the number of originalexpressed RNA. As will be appreciated, although shown as a singleoligonucleotide tethered to the surface of a bead, individual bead caninclude tens to hundreds of thousands or even millions of individualoligonucleotide molecules, where, as noted, the barcode segment can beconstant or relatively constant for a given bead, but where the variableor unique sequence segment will vary across an individual bead. Thisunique molecular sequence segment may include from 5 to about 8 or morenucleotides within the sequence of the oligonucleotides. In some cases,the unique molecular sequence segment can be 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length orlonger. In some cases, the unique molecular sequence segment can be atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 nucleotides in length or longer. In some cases, the unique molecularsequence segment can be at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 nucleotides in length or shorter.

In operation, and with reference to FIGS. 6 and 7, a cell isco-partitioned along with a barcode bearing bead and lysed while thebarcoded oligonucleotides are released from the bead. The poly-T portionof the released barcode oligonucleotide then hybridizes to the poly-Atail of the mRNA. The poly-T segment then primes the reversetranscription of the mRNA to produce a cDNA transcript of the mRNA, butwhich includes each of the sequence segments 708-716 of the barcodeoligonucleotide. Again, because the oligonucleotide 702 includes ananchoring sequence 714, it will more likely hybridize to and primereverse transcription at the sequence end of the poly-A tail of themRNA. Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules will include a common barcode sequence segment710. However, by including the unique random N-mer sequence, thetranscripts made from different mRNA molecules within a given partitionwill vary at this unique sequence. This provides a quantitation featurethat can be identifiable even following any subsequent amplification ofthe contents of a given partition, e.g., the number of unique segmentsassociated with a common barcode can be indicative of the quantity ofmRNA originating from a single partition, and thus, a single cell. Asnoted above, the transcripts are then amplified, cleaned up andsequenced to identify the sequence of the cDNA transcript of the mRNA,as well as to sequence the barcode segment and the unique sequencesegment.

As noted elsewhere herein, while a poly-T primer sequence is described,other targeted or random priming sequences may also be used in primingthe reverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition along withthe contents of the lysed cells, it will be appreciated that in somecases, the gel bead bound oligonucleotides may be used to hybridize adcapture the mRNA on the solid phase of the gel beads, in order tofacilitate the separation of the RNA from other cell contents.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including messenger RNA (mRNA, including mRNA obtained from acell) analysis, is shown in FIG. 9A. As shown, the overalloligonucleotide 902 can be coupled to a bead 904 by a releasable linkage906, such as a disulfide linker. The oligonucleotide may includefunctional sequences that are used in subsequent processing, such asfunctional sequence 908, which may include a sequencer specific flowcell attachment sequence, e.g., a P5 sequence for Illumina sequencingsystems, as well as functional sequence 910, which may includesequencing primer sequences, e.g., a R1 primer binding site for Illuminasequencing systems. A barcode sequence 912 is included within thestructure for use in barcoding the sample RNA. An RNA specific (e.g.,mRNA specific) priming sequence, such as poly-T sequence 914 is alsoincluded in the oligonucleotide structure. An anchoring sequence segment(not shown) may be included to ensure that the poly-T sequencehybridizes at the sequence end of the mRNA. An additional sequencesegment 916 may be provided within the oligonucleotide sequence. Thisadditional sequence can provide a unique molecular sequence segment,e.g., as a random N-mer sequence that varies across individualoligonucleotides coupled to a single bead, whereas barcode sequence 912can be constant among oligonucleotides tethered to an individual bead.As described elsewhere herein, this unique sequence can serve to providea unique identifier of the starting mRNA molecule that was captured, inorder to allow quantitation of the number of original expressed RNA,e.g., mRNA counting. As will be appreciated, although shown as a singleoligonucleotide tethered to the surface of a bead, individual beads caninclude tens to hundreds of thousands or even millions of individualoligonucleotide molecules, where, as noted, the barcode segment can beconstant or relatively constant for a given bead, but where the variableor unique sequence segment will vary across an individual bead.

In an example method of cellular RNA (e.g., mRNA) analysis and inreference to FIG. 9A, a cell is co-partitioned along with a barcodebearing bead, switch oligo 924, and other reagents such as reversetranscriptase, a reducing agent and dNTPs into a partition (e.g., adroplet in an emulsion). In operation 950, the cell is lysed while thebarcoded oligonucleotides 902 are released from the bead (e.g., via theaction of the reducing agent) and the poly-T segment 914 of the releasedbarcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920that is released from the cell. Next, in operation 952 the poly-Tsegment 914 is extended in a reverse transcription reaction using themRNA as a template to produce a cDNA transcript 922 complementary to themRNA and also includes each of the sequence segments 908, 912, 910, 916and 914 of the barcode oligonucleotide. Terminal transferase activity ofthe reverse transcriptase can add additional bases to the cDNAtranscript (e.g., polyC). The switch oligo 924 may then hybridize withthe additional bases added to the cDNA transcript and facilitatetemplate switching. A sequence complementary to the switch oligosequence can then be incorporated into the cDNA transcript 922 viaextension of the cDNA transcript 922 using the switch oligo 924 as atemplate. Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules will include a common barcode sequence segment912. However, by including the unique random N-mer sequence 916, thetranscripts made from different mRNA molecules within a given partitionwill vary at this unique sequence. As described elsewhere herein, thisprovides a quantitation feature that can be identifiable even followingany subsequent amplification of the contents of a given partition, e.g.,the number of unique segments associated with a common barcode can beindicative of the quantity of mRNA originating from a single partition,and thus, a single cell. Following operation 952, the cDNA transcript922 is then amplified with primers 926 (e.g., PCR primers) in operation954. Next, the amplified product is then purified (e.g., via solid phasereversible immobilization (SPRI)) in operation 956. At operation 958,the amplified product is then sheared, ligated to additional functionalsequences, and further amplified (e.g., via PCR). The functionalsequences may include a sequencer specific flow cell attachment sequence930, e.g., a P7 sequence for Illumina sequencing systems, as well asfunctional sequence 928, which may include a sequencing primer bindingsite, e.g., for a R2 primer for Illumina sequencing systems, as well asfunctional sequence 932, which may include a sample index, e.g., an i7sample index sequence for Illumina sequencing systems. In some cases,operations 950 and 952 can occur in the partition, while operations 954,956 and 958 can occur in bulk solution (e.g., in a pooled mixtureoutside of the partition). In the case where a partition is a droplet inan emulsion, the emulsion can be broken and the contents of the dropletpooled in order to complete operations 954, 956 and 958. In some cases,operation 954 may be completed in the partition. In some cases, barcodeoligonucleotides may be digested with exonucleases after the emulsion isbroken. Exonuclease activity can be inhibited byethylenediaminetetraacetic acid (EDTA) following primer digestion.Although described in terms of specific sequence references used forcertain sequencing systems, e.g., Illumina systems, it will beunderstood that the reference to these sequences is for illustrationpurposes only, and the methods described herein may be configured foruse with other sequencing systems incorporating specific priming,attachment, index, and other operational sequences used in thosesystems, e.g., systems available from Ion Torrent, Oxford Nanopore,Genia, Pacific Biosciences, Complete Genomics, and the like.

In an alternative example of a barcode oligonucleotide for use in RNA(e.g., cellular RNA) analysis as shown in FIG. 9A, functional sequence908 may be a P7 sequence and functional sequence 910 may be a R2 primerbinding site. Moreover, the functional sequence 930 may be a P5sequence, functional sequence 928 may be a R1 primer binding site, andfunctional sequence 932 may be an i5 sample index sequence for Illuminasequencing systems. The configuration of the constructs generated bysuch a barcode oligonucleotide can help minimize (or avoid) sequencingof the poly-T sequence during sequencing.

Shown in FIG. 9B is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs intoa partition (e.g., a droplet in an emulsion). The switch oligo 924 maybe labeled with an additional tag 934, e.g. biotin. In operation 951,the cell is lysed while the barcoded oligonucleotides 902 (e.g., asshown in FIG. 9A) are released from the bead (e.g., via the action ofthe reducing agent). In some cases, sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site. In other cases, sequence 908is a P5 sequence and sequence 910 is a R1 primer binding site. Next, thepoly-T segment 914 of the released barcode oligonucleotide hybridizes tothe poly-A tail of mRNA 920 that is released from the cell. In operation953, the poly-T segment 914 is then extended in a reverse transcriptionreaction using the mRNA as a template to produce a cDNA transcript 922complementary to the mRNA and also includes each of the sequencesegments 908, 912, 910, 916 and 914 of the barcode oligonucleotide.Terminal transferase activity of the reverse transcriptase can addadditional bases to the cDNA transcript (e.g., polyC). The switch oligo924 may then hybridize with the cDNA transcript and facilitate templateswitching. A sequence complementary to the switch oligo sequence canthen be incorporated into the cDNA transcript 922 via extension of thecDNA transcript 922 using the switch oligo 924 as a template. Next, anisolation operation 960 can be used to isolate the cDNA transcript 922from the reagents and oligonucleotides in the partition. The additionaltag 934, e.g. biotin, can be contacted with an interacting tag 936,e.g., streptavidin, which may be attached to a magnetic bead 938. Atoperation 960 the cDNA can be isolated with a pull-down operation (e.g.,via magnetic separation, centrifugation) before amplification (e.g., viaPCR) in operation 955, followed by purification (e.g., via solid phasereversible immobilization (SPRI)) in operation 957 and furtherprocessing (shearing, ligation of sequences 928, 932 and 930 andsubsequent amplification (e.g., via PCR)) in operation 959. In somecases where sequence 908 is a P7 sequence and sequence 910 is a R2primer binding site, sequence 930 is a P5 sequence and sequence 928 is aR1 primer binding site and sequence 932 is an i5 sample index sequence.In some cases where sequence 908 is a P5 sequence and sequence 910 is aR1 primer binding site, sequence 930 is a P7 sequence and sequence 928is a R2 primer binding site and sequence 932 is an i7 sample indexsequence. In some cases, as shown, operations 951 and 953 can occur inthe partition, while operations 960, 955, 957 and 959 can occur in bulksolution (e.g., in a pooled mixture outside of the partition). In thecase where a partition is a droplet in an emulsion, the emulsion can bebroken and the contents of the droplet pooled in order to completeoperation 960. The operations 955, 957, and 959 can then be carried outfollowing operation 960 after the transcripts are pooled for processing.

Shown in FIG. 9C is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs in apartition (e.g., a droplet in an emulsion). In operation 961, the cellis lysed while the barcoded oligonucleotides 902 (e.g., as shown in FIG.9A) are released from the bead (e.g., via the action of the reducingagent). In some cases, sequence 908 is a P7 sequence and sequence 910 isa R2 primer binding site. In other cases, sequence 908 is a P5 sequenceand sequence 910 is a R1 primer binding site. Next, the poly-T segment914 of the released barcode oligonucleotide then hybridizes to thepoly-A tail of mRNA 920 that is released from the cell. Next, inoperation 963 the poly-T segment 914 is then extended in a reversetranscription reaction using the mRNA as a template to produce a cDNAtranscript 922 complementary to the mRNA and also includes each of thesequence segments 908, 912, 910, 916 and 914 of the barcodeoligonucleotide. Terminal transferase activity of the reversetranscriptase can add additional bases to the cDNA transcript (e.g.,polyC). The switch oligo 924 may then hybridize with the cDNA transcriptand facilitate template switching. A sequence complementary to theswitch oligo sequence can then be incorporated into the cDNA transcript922 via extension of the cDNA transcript 922 using the switch oligo 924as a template. Following operation 961 and operation 963, mRNA 920 andcDNA transcript 922 are denatured in operation 962. At operation 964, asecond strand is extended from a primer 940 having an additional tag942, e.g. biotin, and hybridized to the cDNA transcript 922. Also inoperation 964, the biotin labeled second strand can be contacted with aninteracting tag 936, e.g. streptavidin, which may be attached to amagnetic bead 938. The cDNA can be isolated with a pull-down operation(e.g., via magnetic separation, centrifugation) before amplification(e.g., via polymerase chain reaction (PCR)) in operation 965, followedby purification (e.g., via solid phase reversible immobilization (SPRI))in operation 967 and further processing (shearing, ligation of sequences928, 932 and 930 and subsequent amplification (e.g., via PCR)) inoperation 969. In some cases where sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site, sequence 930 is a P5 sequenceand sequence 928 is a R1 primer binding site and sequence 932 is an i5sample index sequence. In some cases where sequence 908 is a P5 sequenceand sequence 910 is a R1 primer binding site, sequence 930 is a P7sequence and sequence 928 is a R2 primer binding site and sequence 932is an i7 sample index sequence. In some cases, operations 961 and 963can occur in the partition, while operations 962, 964, 965, 967, and 969can occur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled in order to complete operations 962,964, 965, 967 and 969.

Shown in FIG. 9D is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs. Inoperation 971, the cell is lysed while the barcoded oligonucleotides 902(e.g., as shown in FIG. 9A) are released from the bead (e.g., via theaction of the reducing agent). In some cases, sequence 908 is a P7sequence and sequence 910 is a R2 primer binding site. In other cases,sequence 908 is a P5 sequence and sequence 910 is a R1 primer bindingsite. Next the poly-T segment 914 of the released barcodeoligonucleotide then hybridizes to the poly-A tail of mRNA 920 that isreleased from the cell. Next in operation 973, the poly-T segment 914 isthen extended in a reverse transcription reaction using the mRNA as atemplate to produce a cDNA transcript 922 complementary to the mRNA andalso includes each of the sequence segments 908, 912, 910, 916 and 914of the barcode oligonucleotide. Terminal transferase activity of thereverse transcriptase can add additional bases to the cDNA transcript(e.g., polyC). The switch oligo 924 may then hybridize with the cDNAtranscript and facilitate template switching. A sequence complementaryto the switch oligo sequence can then be incorporated into the cDNAtranscript 922 via extension of the cDNA transcript 922 using the switcholigo 924 as a template. In operation 966, the mRNA 920, cDNA transcript922 and switch oligo 924 can be denatured, and the cDNA transcript 922can be hybridized with a capture oligonucleotide 944 labeled with anadditional tag 946, e.g. biotin. In this operation, the biotin-labeledcapture oligonucleotide 944, which is hybridized to the cDNA transcript,can be contacted with an interacting tag 936, e.g. streptavidin, whichmay be attached to a magnetic bead 938. Following separation from otherspecies (e.g., excess barcoded oligonucleotides) using a pull-downoperation (e.g., via magnetic separation, centrifugation), the cDNAtranscript can be amplified (e.g., via PCR) with primers 926 atoperation 975, followed by purification (e.g., via solid phasereversible immobilization (SPRI)) in operation 977 and furtherprocessing (shearing, ligation of sequences 928, 932 and 930 andsubsequent amplification (e.g., via PCR)) in operation 979. In somecases where sequence 908 is a P7 sequence and sequence 910 is a R2primer binding site, sequence 930 is a P5 sequence and sequence 928 is aR1 primer binding site and sequence 932 is an i5 sample index sequence.In other cases where sequence 908 is a P5 sequence and sequence 910 is aR1 primer binding site, sequence 930 is a P7 sequence and sequence 928is a R2 primer binding site and sequence 932 is an i7 sample indexsequence. In some cases, operations 971 and 973 can occur in thepartition, while operations 966, 975, 977 (purification), and 979 canoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled in order to complete operations 966,975, 977 and 979.

Shown in FIG. 9E is another example method for RNA analysis, includingcellular RNA analysis. In this method, an individual cell isco-partitioned along with a barcode bearing bead, a switch oligo 990,and other reagents such as reverse transcriptase, a reducing agent anddNTPs into a partition (e.g., a droplet in an emulsion). In operation981, the cell is lysed while the barcoded oligonucleotides (e.g., 902 asshown in FIG. 9A) are released from the bead (e.g., via the action ofthe reducing agent). In some cases, sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site. In other cases, sequence 908is a P5 sequence and sequence 910 is a R1 primer binding site. Next, thepoly-T segment of the released barcode oligonucleotide then hybridizesto the poly-A tail of mRNA 920 released from the cell. Next at operation983, the poly-T segment is then extended in a reverse transcriptionreaction to produce a cDNA transcript 922 complementary to the mRNA andalso includes each of the sequence segments 908, 912, 910, 916 and 914of the barcode oligonucleotide. Terminal transferase activity of thereverse transcriptase can add additional bases to the cDNA transcript(e.g., polyC). The switch oligo 990 may then hybridize with the cDNAtranscript and facilitate template switching. A sequence complementaryto the switch oligo sequence and including a T7 promoter sequence, canbe incorporated into the cDNA transcript 922. At operation 968, a secondstrand is synthesized and at operation 970 the T7 promoter sequence canbe used by T7 polymerase to produce RNA transcripts in in vitrotranscription. At operation 985 the RNA transcripts can be purified(e.g., via solid phase reversible immobilization (SPRI)), reversetranscribed to form DNA transcripts, and a second strand can besynthesized for each of the DNA transcripts. In some cases, prior topurification, the RNA transcripts can be contacted with a DNase (e.g.,DNAase I) to break down residual DNA. At operation 987 the DNAtranscripts are then fragmented and ligated to additional functionalsequences, such as sequences 928, 932 and 930 and, in some cases,further amplified (e.g., via PCR). In some cases where sequence 908 is aP7 sequence and sequence 910 is a R2 primer binding site, sequence 930is a P5 sequence and sequence 928 is a R1 primer binding site andsequence 932 is an i5 sample index sequence. In some cases wheresequence 908 is a P5 sequence and sequence 910 is a R1 primer bindingsite, sequence 930 is a P7 sequence and sequence 928 is a R2 primerbinding site and sequence 932 is an i7 sample index sequence. In somecases, prior to removing a portion of the DNA transcripts, the DNAtranscripts can be contacted with an RNase to break down residual RNA.In some cases, operations 981 and 983 can occur in the partition, whileoperations 968, 970, 985 and 987 can occur in bulk (e.g., outside thepartition). In the case where a partition is a droplet in an emulsion,the emulsion can be broken and the contents of the droplet pooled inorder to complete operations 968, 970, 985 and 987.

Another example of a barcode oligonucleotide for use in RNA analysis,including messenger RNA (mRNA, including mRNA obtained from a cell)analysis is shown in FIG. 10. As shown, the overall oligonucleotide 1002is coupled to a bead 1004 by a releasable linkage 1006, such as adisulfide linker. The oligonucleotide may include functional sequencesthat are used in subsequent processing, such as functional sequence1008, which may include a sequencer specific flow cell attachmentsequence, e.g., a P7 sequence, as well as functional sequence 1010,which may include sequencing primer sequences, e.g., a R2 primer bindingsite. A barcode sequence 1012 is included within the structure for usein barcoding the sample RNA. An RNA specific (e.g., mRNA specific)priming sequence, such as poly-T sequence 1014 may be included in theoligonucleotide structure. An anchoring sequence segment (not shown) maybe included to ensure that the poly-T sequence hybridizes at thesequence end of the mRNA. An additional sequence segment 1016 may beprovided within the oligonucleotide sequence. This additional sequencecan provide a unique molecular sequence segment, as described elsewhereherein. An additional functional sequence 1020 may be included for invitro transcription, e.g., a T7 RNA polymerase promoter sequence. Aswill be appreciated, although shown as a single oligonucleotide tetheredto the surface of a bead, individual beads can include tens to hundredsof thousands or even millions of individual oligonucleotide molecules,where, as noted, the barcode segment can be constant or relativelyconstant for a given bead, but where the variable or unique sequencesegment will vary across an individual bead.

In an example method of cellular RNA analysis and in reference to FIG.10, a cell is co-partitioned along with a barcode bearing bead, andother reagents such as reverse transcriptase, reducing agent and dNTPsinto a partition (e.g., a droplet in an emulsion). In operation 1050,the cell is lysed while the barcoded oligonucleotides 1002 are released(e.g., via the action of the reducing agent) from the bead, and thepoly-T segment 1014 of the released barcode oligonucleotide thenhybridizes to the poly-A tail of mRNA 1020. Next at operation 1052, thepoly-T segment is then extended in a reverse transcription reactionusing the mRNA as template to produce a cDNA transcript 1022 of the mRNAand also includes each of the sequence segments 1020, 1008, 1012, 1010,1016, and 1014 of the barcode oligonucleotide. Within any givenpartition, all of the cDNA transcripts of the individual mRNA moleculeswill include a common barcode sequence segment 1012. However, byincluding the unique random N-mer sequence, the transcripts made fromdifferent mRNA molecules within a given partition will vary at thisunique sequence. As described elsewhere herein, this provides aquantitation feature that can be identifiable even following anysubsequent amplification of the contents of a given partition, e.g., thenumber of unique segments associated with a common barcode can beindicative of the quantity of mRNA originating from a single partition,and thus, a single cell. At operation 1054 a second strand issynthesized and at operation 1056 the Tab 7 promoter sequence can beused by T7 polymerase to produce RNA transcripts in in vitrotranscription. At operation 1058 the transcripts are fragmented (e.g.,sheared), ligated to additional functional sequences, and reversetranscribed. The functional sequences may include a sequencer specificflow cell attachment sequence 1030, e.g., a P5 sequence, as well asfunctional sequence 1028, which may include sequencing primers, e.g., aR1 primer binding sequence, as well as functional sequence 1032, whichmay include a sample index, e.g., an i5 sample index sequence. Atoperation 1060 the RNA transcripts can be reverse transcribed to DNA,the DNA amplified (e.g., via PCR), and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the unique sequence segment. In some cases,operations 1050 and 1052 can occur in the partition, while operations1054, 1056, 1058 and 1060 can occur in bulk (e.g., outside thepartition). In the case where a partition is a droplet in an emulsion,the emulsion can be broken and the contents of the droplet pooled inorder to complete operations 1054, 1056, 1058 and 1060.

In an alternative example of a barcode oligonucleotide for use in RNA(e.g., cellular RNA) analysis as shown in FIG. 10, functional sequence1008 may be a P5 sequence and functional sequence 1010 may be a R1primer binding site. Moreover, the functional sequence 1030 may be a P7sequence, functional sequence 1028 may be a R2 primer binding site, andfunctional sequence 1032 may be an i7 sample index sequence.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including messenger RNA (mRNA, including mRNA obtained from acell) analysis is shown in FIG. 11. As shown, the overalloligonucleotide 1102 is coupled to a bead 1104 by a releasable linkage1106, such as a disulfide linker. The oligonucleotide may includefunctional sequences that are used in subsequent processing, such asfunctional sequence 1108, which may include a sequencer specific flowcell attachment sequence, e.g., a P5 sequence, as well as functionalsequence 1110, which may include sequencing primer sequences, e.g., a R1primer binding site. In some cases, sequence 1108 is a P7 sequence andsequence 1110 is a R2 primer binding site. A barcode sequence 1112 isincluded within the structure for use in barcoding the sample RNA. Anadditional sequence segment 1116 may be provided within theoligonucleotide sequence. In some cases, this additional sequence canprovide a unique molecular sequence segment, as described elsewhereherein. An additional sequence 1114 may be included to facilitatetemplate switching, e.g., polyG. As will be appreciated, although shownas a single oligonucleotide tethered to the surface of a bead,individual beads can include tens to hundreds of thousands or evenmillions of individual oligonucleotide molecules, where, as noted, thebarcode segment can be constant or relatively constant for a given bead,but where the variable or unique sequence segment will vary across anindividual bead.

In an example method of cellular mRNA analysis and in reference to FIG.11, a cell is co-partitioned along with a barcode bearing bead, poly-Tsequence, and other reagents such as reverse transcriptase, a reducingagent and dNTPs into a partition (e.g., a droplet in an emulsion). Inoperation 1150, the cell is lysed while the barcoded oligonucleotidesare released from the bead (e.g., via the action of the reducing agent)and the poly-T sequence hybridizes to the poly-A tail of mRNA 1120released from the cell. Next, in operation 1152, the poly-T sequence isthen extended in a reverse transcription reaction using the mRNA as atemplate to produce a cDNA transcript 1122 complementary to the mRNA.Terminal transferase activity of the reverse transcriptase can addadditional bases to the cDNA transcript (e.g., polyC). The additionalbases added to the cDNA transcript, e.g., polyC, can then to hybridizewith 1114 of the barcoded oligonucleotide. This can facilitate templateswitching and a sequence complementary to the barcode oligonucleotidecan be incorporated into the cDNA transcript. The transcripts can befurther processed (e.g., amplified, portions removed, additionalsequences added, etc.) and characterized as described elsewhere herein,e.g., by sequencing. The configuration of the constructs generated bysuch a method can help minimize (or avoid) sequencing of the poly-Tsequence during sequencing.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including cellular RNA analysis is shown in FIG. 12A. Asshown, the overall oligonucleotide 1202 is coupled to a bead 1204 by areleasable linkage 1206, such as a disulfide linker. The oligonucleotidemay include functional sequences that are used in subsequent processing,such as functional sequence 1208, which may include a sequencer specificflow cell attachment sequence, e.g., a P5 sequence, as well asfunctional sequence 1210, which may include sequencing primer sequences,e.g., a R1 primer binding site. In some cases, sequence 1208 is a P7sequence and sequence 1210 is a R2 primer binding site. A barcodesequence 1212 is included within the structure for use in barcoding thesample RNA. An additional sequence segment 1216 may be provided withinthe oligonucleotide sequence. In some cases, this additional sequencecan provide a unique molecular sequence segment, as described elsewhereherein. As will be appreciated, although shown as a singleoligonucleotide tethered to the surface of a bead, individual beads caninclude tens to hundreds of thousands or even millions of individualoligonucleotide molecules, where, as noted, the barcode segment can beconstant or relatively constant for a given bead, but where the variableor unique sequence segment will vary across an individual bead. In anexample method of cellular RNA analysis using this barcode, a cell isco-partitioned along with a barcode bearing bead and other reagents suchas RNA ligase and a reducing agent into a partition (e.g. a droplet inan emulsion). The cell is lysed while the barcoded oligonucleotides arereleased (e.g., via the action of the reducing agent) from the bead. Thebarcoded oligonucleotides can then be ligated to the 5′ end of mRNAtranscripts while in the partitions by RNA ligase. Subsequent operationsmay include purification (e.g., via solid phase reversibleimmobilization (SPRI)) and further processing (shearing, ligation offunctional sequences, and subsequent amplification (e.g., via PCR)), andthese operations may occur in bulk (e.g., outside the partition). In thecase where a partition is a droplet in an emulsion, the emulsion can bebroken and the contents of the droplet pooled for the additionaloperations.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including cellular RNA analysis is shown in FIG. 12B. Asshown, the overall oligonucleotide 1222 is coupled to a bead 1224 by areleasable linkage 1226, such as a disulfide linker. The oligonucleotidemay include functional sequences that are used in subsequent processing,such as functional sequence 1228, which may include a sequencer specificflow cell attachment sequence, e.g., a P5 sequence, as well asfunctional sequence 1230, which may include sequencing primer sequences,e.g., a R1 primer binding site. In some cases, sequence 1228 is a P7sequence and sequence 1230 is a R2 primer binding site. A barcodesequence 1232 is included within the structure for use in barcoding thesample RNA. A priming sequence 1234 (e.g., a random priming sequence)can also be included in the oligonucleotide structure, e.g., a randomhexamer. An additional sequence segment 1236 may be provided within theoligonucleotide sequence. In some cases, this additional sequenceprovides a unique molecular sequence segment, as described elsewhereherein. As will be appreciated, although shown as a singleoligonucleotide tethered to the surface of a bead, individual beads caninclude tens to hundreds of thousands or even millions of individualoligonucleotide molecules, where, as noted, the barcode segment can beconstant or relatively constant for a given bead, but where the variableor unique sequence segment will vary across an individual bead. In anexample method of cellular mRNA analysis using the barcodeoligonucleotide of FIG. 12B, a cell is co-partitioned along with abarcode bearing bead and additional reagents such as reversetranscriptase, a reducing agent and dNTPs into a partition (e.g., adroplet in an emulsion). The cell is lysed while the barcodedoligonucleotides are released from the bead (e.g., via the action of thereducing agent). In some cases, sequence 1228 is a P7 sequence andsequence 1230 is a R2 primer binding site. In other cases, sequence 1228is a P5 sequence and sequence 1230 is a R1 primer binding site. Thepriming sequence 1234 of random hexamers can randomly hybridize cellularmRNA. The random hexamer sequence can then be extended in a reversetranscription reaction using mRNA from the cell as a template to producea cDNA transcript complementary to the mRNA and also includes each ofthe sequence segments 1228, 1232, 1230, 1236, and 1234 of the barcodeoligonucleotide. Subsequent operations may include purification (e.g.,via solid phase reversible immobilization (SPRI)), further processing(shearing, ligation of functional sequences, and subsequentamplification (e.g., via PCR)), and these operations may occur in bulk(e.g., outside the partition). In the case where a partition is adroplet in an emulsion, the emulsion can be broken and the contents ofthe droplet pooled for additional operations. Additional reagents thatmay be co-partitioned along with the barcode bearing bead may includeoligonucleotides to block ribosomal RNA (rRNA) and nucleases to digestgenomic DNA and cDNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing.

The single cell analysis methods described herein may also be useful inthe analysis of the whole transcriptome. Referring back to the barcodeof FIG. 12B, the priming sequence 1234 may be a random N-mer. In somecases, sequence 1228 is a P7 sequence and sequence 1230 is a R2 primerbinding site. In other cases, sequence 1228 is a P5 sequence andsequence 1230 is a R1 primer binding site. In an example method of wholetranscriptome analysis using this barcode, the individual cell isco-partitioned along with a barcode bearing bead, poly-T sequence, andother reagents such as reverse transcriptase, polymerase, a reducingagent and dNTPs into a partition (e.g., droplet in an emulsion). In anoperation of this method, the cell is lysed while the barcodedoligonucleotides are released from the bead (e.g., via the action of thereducing agent) and the poly-T sequence hybridizes to the poly-A tail ofcellular mRNA. In a reverse transcription reaction using the mRNA astemplate, cDNA transcripts of cellular mRNA can be produced. The RNA canthen be degraded with an RNase. The priming sequence 1234 in thebarcoded oligonucleotide can then randomly hybridize to the cDNAtranscripts. The oligonucleotides can be extended using polymeraseenzymes and other extension reagents co-partitioned with the bead andcell similar to as shown in FIG. 3 to generate amplification products(e.g., barcoded fragments), similar to the example amplification productshown in FIG. 3 (panel F). The barcoded nucleic acid fragments may, insome cases subjected to further processing (e.g., amplification,addition of additional sequences, clean up processes, etc. as describedelsewhere herein) characterized, e.g., through sequence analysis. Inthis operation, sequencing signals can come from full length RNA.

Although operations with various barcode designs have been discussedindividually, individual beads can include barcode oligonucleotides ofvarious designs for simultaneous use.

In addition to characterizing individual cells or cell sub-populationsfrom larger populations, the processes and systems described herein mayalso be used to characterize individual cells as a way to provide anoverall profile of a cellular, or other organismal population. A varietyof applications require the evaluation of the presence andquantification of different cell or organism types within a populationof cells, including, for example, microbiome analysis andcharacterization, environmental testing, food safety testing,epidemiological analysis, e.g., in tracing contamination or the like. Inparticular, the analysis processes described above may be used toindividually characterize, sequence and/or identify large numbers ofindividual cells within a population. This characterization may then beused to assemble an overall profile of the originating population, whichcan provide important prognostic and diagnostic information.

For example, shifts in human microbiomes, including, e.g., gut, buccal,epidermal microbiomes, etc., have been identified as being bothdiagnostic and prognostic of different conditions or general states ofhealth. Using the single cell analysis methods and systems describedherein, one can again, characterize, sequence and identify individualcells in an overall population, and identify shifts within thatpopulation that may be indicative of diagnostic ally relevant factors.By way of example, sequencing of bacterial 16S ribosomal RNA genes hasbeen used as a highly accurate method for taxonomic classification ofbacteria. Using the targeted amplification and sequencing processesdescribed above can provide identification of individual cells within apopulation of cells. One may further quantify the numbers of differentcells within a population to identify current states or shifts in statesover time. See, e.g., Morgan et al, PLoS Comput. Biol., Ch. 12, December2012, 8(12):e1002808, and Ram et al., Syst. Biol. Reprod. Med., June2011, 57(3): 162-170, each of which is incorporated herein by referencein its entirety for all purposes. Likewise, identification and diagnosisof infection or potential infection may also benefit from the singlecell analyses described herein, e.g., to identify microbial speciespresent in large mixes of other cells or other biological material,cells and/or nucleic acids, including the environments described above,as well as any other diagnostically relevant environments, e.g.,cerebrospinal fluid, blood, fecal or intestinal samples, or the like.

The foregoing analyses may also be particularly useful in thecharacterization of potential drug resistance of different cells, e.g.,cancer cells, bacterial pathogens, etc., through the analysis ofdistribution and profiling of different resistance markers/mutationsacross cell populations in a given sample. Additionally,characterization of shifts in these markers/mutations across populationsof cells over time can provide valuable insight into the progression,alteration, prevention, and treatment of a variety of diseasescharacterized by such drug resistance issues.

Although described in terms of cells, it will be appreciated that any ofa variety of individual biological organisms, or components of organismsare encompassed within this description, including, for example, cells,viruses, organelles, cellular inclusions, vesicles, or the like.Additionally, where referring to cells, it will be appreciated that suchreference includes any type of cell, including without limitationprokaryotic cells, eukaryotic cells, bacterial, fungal, plant,mammalian, or other animal cell types, mycoplasmas, normal tissue cells,tumor cells, or any other cell type, whether derived from single cell ormulticellular organisms.

Similarly, analysis of different environmental samples to profile themicrobial organisms, viruses, or other biological contaminants that arepresent within such samples, can provide important information aboutdisease epidemiology, and potentially aid in forecasting diseaseoutbreaks, epidemics an pandemics.

As described above, the methods, systems and compositions describedherein may also be used for analysis and characterization of otheraspects of individual cells or populations of cells. In one exampleprocess, a sample is provided that contains cells that are to beanalyzed and characterized as to their cell surface proteins. Alsoprovided is a library of antibodies, antibody fragments, or othermolecules having a binding affinity to the cell surface proteins orantigens (or other cell features) for which the cell is to becharacterized (also referred to herein as cell surface feature bindinggroups). For ease of discussion, these affinity groups are referred toherein as binding groups. The binding groups can include a reportermolecule that is indicative of the cell surface feature to which thebinding group binds. In particular, a binding group type that isspecific to one type of cell surface feature will comprise a firstreporter molecule, while a binding group type that is specific to adifferent cell surface feature will have a different reporter moleculeassociated with it. In some aspects, these reporter molecules willcomprise oligonucleotide sequences. Oligonucleotide based reportermolecules provide advantages of being able to generate significantdiversity in terms of sequence, while also being readily attachable tomost biomolecules, e.g., antibodies, etc., as well as being readilydetected, e.g., using sequencing or array technologies. In the exampleprocess, the binding groups include oligonucleotides attached to them.Thus, a first binding group type, e.g., antibodies to a first type ofcell surface feature, will have associated with it a reporteroligonucleotide that has a first nucleotide sequence. Different bindinggroup types, e.g., antibodies having binding affinity for other,different cell surface features, will have associated therewith reporteroligonucleotides that comprise different nucleotide sequences, e.g.,having a partially or completely different nucleotide sequence. In somecases, for each type of cell surface feature binding group, e.g.,antibody or antibody fragment, the reporter oligonucleotide sequence maybe known and readily identifiable as being associated with the knowncell surface feature binding group. These oligonucleotides may bedirectly coupled to the binding group, or they may be attached to abead, molecular lattice, e.g., a linear, globular, cross-slinked, orother polymer, or other framework that is attached or otherwiseassociated with the binding group, which allows attachment of multiplereporter oligonucleotides to a single binding group.

In the case of multiple reporter molecules coupled to a single bindinggroup, such reporter molecules can comprise the same sequence, or aparticular binding group will include a known set of reporteroligonucleotide sequences. As between different binding groups, e.g.,specific for different cell surface features, the reporter molecules canbe different and attributable to the particular binding group.

Attachment of the reporter groups to the binding groups may be achievedthrough any of a variety of direct or indirect, covalent or non-covalentassociations or attachments. For example, in the case of oligonucleotidereporter groups associated with antibody based binding groups, sucholigonucleotides may be covalently attached to a portion of an antibodyor antibody fragment using chemical conjugation techniques (e.g.,Lightning-Link® antibody labeling kits available from InnovaBiosciences), as well as other non-covalent attachment mechanisms, e.g.,using biotinylated antibodies and oligonucleotides (or beads thatinclude one or more biotinylated linker, coupled to oligonucleotides)with an avidin or streptavidin linker. Antibody and oligonucleotidebiotinylation techniques are available (See, e.g., Fang, et al.,Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labeling andAffinity Purification of Synthetic Oligonucleotides, Nucleic Acids Res.Jan. 15, 2003; 31(2):708-715, DNA 3′ End Biotinylation Kit, availablefrom Thermo Scientific, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes). Likewise,protein and peptide biotinylation techniques have been developed and arereadily available (See, e.g., U.S. Pat. No. 6,265,552, the fulldisclosures of which are incorporated herein by reference in theirentirety for all purposes).

The reporter oligonucleotides may be provided having any of a range ofdifferent lengths, depending upon the diversity of reporter moleculesdesired or a given analysis, the sequence detection scheme employed, andthe like. In some cases, these reporter sequences can be greater thanabout 5 nucleotides in length, greater than about 10 nucleotides inlength, greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150or even 200 nucleotides in length. In some cases, these reporternucleotides may be less than about 250 nucleotides in length, less thanabout 200, 180, 150, 120 100, 90, 80, 70, 60, 50, 40, or even 30nucleotides in length. In many cases, the reporter oligonucleotides maybe selected to provide barcoded products that are already sized, andotherwise configured to be analyzed on a sequencing system. For example,these sequences may be provided at a length that ideally createssequenceable products of a desired length for particular sequencingsystems. Likewise, these reporter oligonucleotides may includeadditional sequence elements, in addition to the reporter sequence, suchas sequencer attachment sequences, sequencing primer sequences,amplification primer sequences, or the complements to any of these.

In operation, a cell-containing sample is incubated with the bindingmolecules and their associated reporter oligonucleotides, for any of thecell surface features desired to be analyzed. Following incubation, thecells are washed to remove unbound binding groups. Following washing,the cells are partitioned into separate partitions, e.g., droplets,along with the barcode carrying beads described above, where eachpartition includes a limited number of cells, e.g., in some cases, asingle cell. Upon releasing the barcodes from the beads, they will primethe amplification and barcoding of the reporter oligonucleotides. Asnoted above, the barcoded replicates of the reporter molecules mayadditionally include functional sequences, such as primer sequences,attachment sequences or the like.

The barcoded reporter oligonucleotides are then subjected to sequenceanalysis to identify which reporter oligonucleotides bound to the cellswithin the partitions. Further, by also sequencing the associatedbarcode sequence, one can identify that a given cell surface featurelikely came from the same cell as other, different cell surfacefeatures, whose reporter sequences include the same barcode sequence,i.e., they were derived from the same partition.

Based upon the reporter molecules that emanate from an individualpartition based upon the presence of the barcode sequence, one may thencreate a cell surface profile of individual cells from a population ofcells. Profiles of individual cells or populations of cells may becompared to profiles from other cells, e.g., ‘normal’ cells, to identifyvariations in cell surface features, which may provide diagnosticallyrelevant information. In particular, these profiles may be particularlyuseful in the diagnosis of a variety of disorders that are characterizedby variations in cell surface receptors, such as cancer and otherdisorders.

V. Devices and Systems

Also provided herein are the microfluidic devices used for partitioningthe cells as described above. Such microfluidic devices can comprisechannel networks for carrying out the partitioning process like thoseset forth in FIGS. 1 and 2. Examples of particularly useful microfluidicdevices are described in U.S. Provisional Patent Application No.61/977,804, filed Apr. 4, 2014, and incorporated herein by reference inits entirety for all purposes. Briefly, these microfluidic devices cancomprise channel networks, such as those described herein, forpartitioning cells into separate partitions, and co-partitioning suchcells with oligonucleotide barcode library members, e.g., disposed onbeads. These channel networks can be disposed within a solid body, e.g.,a glass, semiconductor or polymer body structure in which the channelsare defined, where those channels communicate at their termini withreservoirs for receiving the various input fluids, and for the ultimatedeposition of the partitioned cells, etc., from the output of thechannel networks. By way of example, and with reference to FIG. 2, areservoir fluidly coupled to channel 202 may be provided with an aqueoussuspension of cells 214, while a reservoir coupled to channel 204 may beprovided with an aqueous suspension of beads 216 carrying theoligonucleotides. Channel segments 206 and 208 may be provided with anon-aqueous solution, e.g., an oil, into which the aqueous fluids arepartitioned as droplets at the channel junction 212. Finally, an outletreservoir may be fluidly coupled to channel 210 into which thepartitioned cells and beads can be delivered and from which they may beharvested. As will be appreciated, while described as reservoirs, itwill be appreciated that the channel segments may be coupled to any of avariety of different fluid sources or receiving components, includingtubing, manifolds, or fluidic components of other systems.

Also provided are systems that control flow of these fluids through thechannel networks e.g., through applied pressure differentials,centrifugal force, electrokinetic pumping, capillary or gravity flow, orthe like.

VI. Kits

Also provided herein are kits for analyzing individual cells or smallpopulations of cells. The kits may include one, two, three, four, fiveor more, up to all of partitioning fluids, including both aqueousbuffers and non-aqueous partitioning fluids or oils, nucleic acidbarcode libraries that are releasably associated with beads, asdescribed herein, microfluidic devices, reagents for disrupting cellsamplifying nucleic acids, and providing additional functional sequenceson fragments of cellular nucleic acids or replicates thereof, as well asinstructions for using any of the foregoing in the methods describedherein.

VII. Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 17 shows acomputer system 1701 that is programmed or otherwise configured toimplement methods of the disclosure including nucleic acid sequencingmethods, interpretation of nucleic acid sequencing data and analysis ofcellular nucleic acids, such as RNA (e.g., mRNA), and characterizationof cells from sequencing data. The computer system 1701 can be anelectronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 1701 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1705, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1701 also includes memory or memorylocation 1710 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1715 (e.g., hard disk), communicationinterface 1720 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1725, such as cache, othermemory, data storage and/or electronic display adapters. The memory1710, storage unit 1715, interface 1720 and peripheral devices 1725 arein communication with the CPU 1705 through a communication bus (solidlines), such as a motherboard. The storage unit 1715 can be a datastorage unit (or data repository) for storing data. The computer system1701 can be operatively coupled to a computer network (“network”) 1730with the aid of the communication interface 1720. The network 1730 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1730 insome cases is a telecommunication and/or data network. The network 1730can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1730, in some cases withthe aid of the computer system 1701, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1701 tobehave as a client or a server.

The CPU 1705 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1710. The instructionscan be directed to the CPU 1705, which can subsequently program orotherwise configure the CPU 1705 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1705 can includefetch, decode, execute, and writeback.

The CPU 1705 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1701 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1715 can store files, such as drivers, libraries andsaved programs. The storage unit 1715 can store user data, e.g., userpreferences and user programs. The computer system 1701 in some casescan include one or more additional data storage units that are externalto the computer system 1701, such as located on a remote server that isin communication with the computer system 1701 through an intranet orthe Internet.

The computer system 1701 can communicate with one or more remotecomputer systems through the network 1730. For instance, the computersystem 1701 can communicate with a remote computer system of a user.Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), or personal digital assistants. The user canaccess the computer system 1701 via the network 1730.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1701, such as, for example, on thememory 1710 or electronic storage unit 1715. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1705. In some cases, thecode can be retrieved from the storage unit 1715 and stored on thememory 1710 for ready access by the processor 1705. In some situations,the electronic storage unit 1715 can be precluded, andmachine-executable instructions are stored on memory 1710.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1701, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1701 can include or be in communication with anelectronic display 1735 that comprises a user interface (UI) 1740 forproviding, for example, results of nucleic acid sequencing, analysis ofnucleic acid sequencing data, characterization of nucleic acidsequencing samples, cell characterizations, etc. Examples of UI'sinclude, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1705. Thealgorithm can, for example, initiate nucleic acid sequencing, processnucleic acid sequencing data, interpret nucleic acid sequencing results,characterize nucleic acid samples, characterize cells, etc.

VIII. Examples Example I Cellular RNA Analysis Using Emulsions

In an example, reverse transcription with template switching and cDNAamplification (via PCR) is performed in emulsion droplets withoperations as shown in FIG. 9A. The reaction mixture that is partitionedfor reverse transcription and cDNA amplification (via PCR) includes1,000 cells or 10,000 cells or 10 ng of RNA, beads bearing barcodedoligonucleotides/0.2% Tx-100/5× Kapa buffer, 2× Kapa HS HiFi Ready Mix,4 μM switch oligo, and Smartscribe. Where cells are present, the mixtureis partitioned such that a majority or all of the droplets comprise asingle cell and single bead. The cells are lysed while the barcodedoligonucleotides are released from the bead, and the poly-T segment ofthe barcoded oligonucleotide hybridizes to the poly-A tail of mRNA thatis released from the cell as in operation 950. The poly-T segment isextended in a reverse transcription reaction as in operation 952 and thecDNA transcript is amplified as in operation 954. The thermal cyclingconditions are 42° C. for 130 minutes; 98° C. for 2 min; and 35 cyclesof the following 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 6min. Following thermal cycling, the emulsion is broken and thetranscripts are purified with Dynabeads and 0.6×SPRI as in operation956.

The yield from template switch reverse transcription and PCR inemulsions is shown for 1,000 cells in FIG. 13A and 10,000 cells in FIG.13C and 10 ng of RNA in FIG. 13B (Smartscribe line). The cDNAtranscripts from RT and PCR performed in emulsions for 10 ng RNA issheared and ligated to functional sequences, cleaned up with 0.8×SPRI,and is further amplified by PCR as in operation 958. The amplificationproduct is cleaned up with 0.8×SPRI. The yield from this processing isshown in FIG. 13B (SSII line).

Example II Cellular RNA Analysis Using Emulsions

In another example, reverse transcription with template switching andcDNA amplification (via PCR) is performed in emulsion droplets withoperations as shown in FIG. 9A. The reaction mixture that is partitionedfor reverse transcription and cDNA amplification (via PCR) includesJurkat cells, beads bearing barcoded oligonucleotides/0.2%TritonX-100/5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switcholigo, and Smartscribe. The mixture is partitioned such that a majorityor all of the droplets comprise a single cell and single bead. The cellsare lysed while the barcoded oligonucleotides are released from thebead, and the poly-T segment of the barcoded oligonucleotide hybridizesto the poly-A tail of mRNA that is released from the cell as inoperation 950. The poly-T segment is extended in a reverse transcriptionreaction as in operation 952 and the cDNA transcript is amplified as inoperation 954. The thermal cycling conditions are 42° C. for 130minutes; 98° C. for 2 min; and 35 cycles of the following 98° C. for 15sec, 60° C. for 20 sec, and 72° C. for 6 min. Following thermal cycling,the emulsion is broken and the transcripts are cleaned-up with Dynabeadsand 0.6×SPRI as in operation 956. The yield from reactions with variouscell numbers (625 cells, 1,250 cells, 2,500 cells, 5,000 cells, and10,000 cells) is shown in FIG. 14A. These yields are confirmed withGADPH qPCR assay results shown in FIG. 14B.

Example III RNA Analysis Using Emulsions

In another example, reverse transcription is performed in emulsiondroplets and cDNA amplification is performed in bulk in a manner similarto that as shown in FIG. 9C. The reaction mixture that is partitionedfor reverse transcription includes beads bearing barcodedoligonucleotides, 10 ng Jurkat RNA (e.g., Jurkat mRNA), 5× First-Strandbuffer, and Smartscribe. The barcoded oligonucleotides are released fromthe bead, and the poly-T segment of the barcoded oligonucleotidehybridizes to the poly-A tail of the RNA as in operation 961. The poly-Tsegment is extended in a reverse transcription reaction as in operation963. The thermal cycling conditions for reverse transcription are onecycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min.Following thermal cycling, the emulsion is broken and RNA and cDNAtranscripts are denatured as in operation 962. A second strand is thensynthesized by primer extension with a primer having a biotin tag as inoperation 964. The reaction conditions for this primer extension includecDNA transcript as the first strand and biotinylated extension primerranging in concentration from 0.5-3.0 μM. The thermal cycling conditionsare one cycle at 98° C. for 3 min and one cycle of 98° C. for 15 sec,60° C. for 20 sec, and 72° C. for 30 min. Following primer extension,the second strand is pulled down with Dynabeads MyOne Streptavidin C1and T1, and cleaned-up with Agilent SureSelect XT buffers. The secondstrand is pre-amplified via PCR as in operation 965 with the followingcycling conditions—one cycle at 98° C. for 3 min and one cycle of 98° C.for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min. The yield forvarious concentrations of biotinylated primer (0.5 M, 1.0 μM, 2.0 μM,and 3.0 μM) is shown in FIG. 15.

Example IV RNA Analysis Using Emulsions

In another example, in vitro transcription by T7 polymerase is used toproduce RNA transcripts as shown in FIG. 10. The mixture that ispartitioned for reverse transcription includes beads bearing barcodedoligonucleotides which also include a T7 RNA polymerase promotersequence, 10 ng human RNA (e.g., human mRNA), 5× First-Strand buffer,and Smartscribe. The mixture is partitioned such that a majority or allof the droplets comprise a single bead. The barcoded oligonucleotidesare released from the bead, and the poly-T segment of the barcodedoligonucleotide hybridizes to the poly-A tail of the RNA as in operation1050. The poly-T segment is extended in a reverse transcription reactionas in operation 1052. The thermal cycling conditions are one cycle at42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermalcycling, the emulsion is broken and the remaining operations areperformed in bulk. A second strand is then synthesized by primerextension as in operation 1054. The reaction conditions for this primerextension include cDNA transcript as template and extension primer. Thethermal cycling conditions are one cycle at 98° C. for 3 min and onecycle of 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min.Following this primer extension, the second strand is purified with0.6×SPRI. As in operation 1056, in vitro transcription is then performedto produce RNA transcripts. In vitro transcription is performedovernight, and the transcripts are purified with 0.6×SPRI. The RNAyields from in vitro transcription are shown in FIG. 16.

While some embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for generating a sequencing set fornucleic acid sequencing, comprising: (a) bringing an aqueous fluidcomprising a plurality of cells and a plurality of beads in contact witha continuous phase comprising an oil that is immiscible with saidaqueous fluid, to generate a plurality droplets, wherein a given dropletof said plurality of droplets comprises a single cell from saidplurality of cells and a single bead from said plurality of beads, andwherein said single bead comprises nucleic acid molecules eachcomprising a common barcode sequence; (b) in said given droplet,subjecting messenger ribonucleic acid (mRNA) molecules from said singlecell to reverse transcription in the presence of said nucleic acidmolecules to yield complementary deoxyribonucleic acid (cDNA) moleculescomprising said common barcode sequence; (c) breaking said plurality ofdroplets, thereby releasing a plurality of cDNA molecules from saidplurality of droplets, wherein said plurality of cDNA moleculescomprises said cDNA molecules or derivatives thereof; and (d) subjectingsaid plurality of cDNA molecules to one or more reactions to generatesaid sequencing set.
 2. The method of claim 1, wherein said nucleic acidmolecules comprise at least 1,000 nucleic acid molecules.
 3. The methodof claim 1, wherein said nucleic acid molecules comprise at least100,000 nucleic acid molecules.
 4. The method of claim 1, wherein saidplurality of droplets is generated at a flow rate of at least about1,000 Hz.
 5. The method of claim 1, wherein said plurality of beadscomprises a plurality of nucleic acid molecules comprising barcodesequences that are different across said plurality of beads.
 6. Themethod of claim 1, further comprising digesting nucleic acid moleculesfrom said given droplet subsequent to breaking said plurality ofdroplets.
 7. The method of claim 1, further comprising, subsequent to(d), pooling said plurality of cDNA molecules and performing said one ormore reactions in bulk.
 8. The method of claim 1, wherein said singlecell and said single bead are partitioned in said given droplet using achannel structure comprising a first channel in fluid communication witha first source comprising a suspension of said plurality of cells and asecond channel in fluid communication with a second source comprising asuspension of said plurality of beads.
 9. The method of claim 8, whereinsaid second source is in fluid communication with a flow regulator thatprovides a regular flow of said plurality of beads in said secondchannel.
 10. The method of claim 9, wherein said plurality of beads flowin said second channel at a flow rate having a coefficient of variationof less than 30% as determined by number of beads that flow past a givenpoint in said second channel within a one second period of time.
 11. Themethod of claim 10, wherein said coefficient of variation is less than20%.
 12. The method of claim 8, wherein individual cells of saidplurality of cells have sizes that are less than a cross-section of saidfirst channel.
 13. The method of claim 8, wherein individual beads ofsaid plurality of beads have sizes that are less than a cross-section ofsaid second channel.
 14. The method of claim 1, further comprising,prior to (b), releasing said nucleic acid molecules from said singlebead.
 15. The method of claim 14, wherein said nucleic acid moleculesare released from said single bead upon exposure to a chemical stimulusin said droplet.
 16. The method of claim 1, wherein said single bead isa gel bead.
 17. The method of claim 1, wherein said single cell is in apolymer matrix.
 18. The method of claim 1, wherein said continuous phasecomprises a fluorinated oil and a fluorosurfactant that inhibitscoalescence of said plurality of droplets.
 19. The method of claim 1,wherein said one or more reactions comprise nucleic acid amplificationthat generates amplified products from said plurality of cDNA molecules.20. The method of claim 19, further comprising purifying said amplifiedproducts.
 21. The method of claim 19, further comprising ligatingfunctional sequences to said amplified products, wherein said functionalsequences permit attachment of said sequencing set to a flow cell of asequencer for said nucleic acid sequencing.
 22. The method of claim 19,wherein said nucleic acid amplification is polymerase chain reaction.23. The method of claim 1, wherein said one or more reactions compriseaddition of functional sequences to said plurality of cDNA molecules,wherein said functional sequences permit attachment of said sequencingset to a flow cell of a sequencer for nucleic acid sequencing.
 24. Themethod of claim 1, wherein said aqueous fluid comprises said pluralityof beads.
 25. The method of claim 1, further comprising using asequencer to subject said sequencing set to said nucleic acid sequencingto generate a plurality of sequences comprising sequences correspondingto said mRNA molecules and said common barcode sequence.
 26. The methodof claim 1, wherein said plurality of beads have substantiallymonodisperse cross-sectional dimensions.
 27. The method of claim 1,wherein each of said nucleic acid molecules comprises said commonbarcode sequence and a unique molecular sequence, wherein said commonbarcode sequence is constant across said nucleic acid molecules, andwherein said unique molecular sequence varies across said nucleic acidmolecules.
 28. The method of claim 27, wherein each of said nucleic acidmolecules comprises an RNA specific priming sequence.
 29. The method ofclaim 27, wherein said nucleic acid molecules comprise functionalsequences that facilitate sequencing of said sequencing set.
 30. Themethod of claim 1, wherein said plurality of droplets comprisestransposases.